Inverse association between apolipoprotein C-II and cardiovascular mortality: role of lipoprotein lipase activity modulation.

AIMS: Apolipoprotein C-II (ApoC-II) is thought to activate lipoprotein lipase (LPL) and is therefore a possible target for treating hypertriglyceridemia. Its relationship with cardiovascular risk has not been investigated in large-scale epidemiologic studies, particularly allowing for apolipoprotein C-III (ApoC-III), an LPL antagonist. Furthermore, the exact mechanism of ApoC-II-mediated LPL activation is unclear. METHODS AND RESULTS: ApoC-II was measured in 3141 LURIC participants of which 590 died from cardiovascular diseases during a median (inter-quartile range) follow-up of 9.9 (8.7-10.7) years. Apolipoprotein C-II-mediated activation of the glycosylphosphatidylinositol high-density lipoprotein binding protein 1 (GPIHBP1)-LPL complex was studied using enzymatic activity assays with fluorometric lipase and very low-density lipoprotein (VLDL) substrates. The mean ApoC-II concentration was 4.5 (2.4) mg/dL. The relationship of ApoC-II quintiles with cardiovascular mortality exhibited a trend toward an inverse J-shape, with the highest risk in the first (lowest) quintile and lowest risk in the middle quintile. Compared with the first quintile, all other quintiles were associated with decreased cardiovascular mortality after multivariate adjustments including ApoC-III as a covariate (all P < 0.05). In experiments using fluorometric substrate-based lipase assays, there was a bell-shaped relationship for the effect of ApoC-II on GPIHBP1-LPL activity when exogenous ApoC-II was added. In ApoC-II-containing VLDL substrate-based lipase assays, GPIHBP1-LPL enzymatic activity was almost completely blocked by a neutralizing anti-ApoC-II antibody. CONCLUSION: The present epidemiologic data suggest that increasing low circulating ApoC-II levels may reduce cardiovascular risk. This conclusion is supported by the observation that optimal ApoC-II concentrations are required for maximal GPIHBP1-LPL enzymatic activity.

The Monomeric α-Crystallin Domain of the Small Heat-shock Proteins αB-crystallin and Hsp27 Binds Amyloid Fibril Ends.

Small heat-shock proteins (sHSPs) are ubiquitously expressed molecular chaperones present in all kingdoms of life that inhibit protein misfolding and aggregation. Despite their importance in proteostasis, the structure-function relationships of sHSPs remain elusive. Human sHSPs are characterised by a central, highly conserved α-crystallin domain (ACD) and variable-length N- and C-terminal regions. The ACD forms antiparallel homodimers via an extended ß-strand, creating a shared ß-sheet at the dimer interface. The N- and C-terminal regions mediate formation of higher order oligomers that are thought to act as storage forms for chaperone-active dimers. We investigated the interactions of the ACD of two human sHSPs, αB-crystallin (αB-C) and Hsp27, with apolipoprotein C-II amyloid fibrils using analytical ultracentrifugation and nuclear magnetic resonance spectroscopy. The ACD was found to interact transiently with amyloid fibrils to inhibit fibril elongation and naturally occurring fibril end-to-end joining. This interaction was sensitive to the concentration of fibril ends indicating a 'fibril-capping' interaction. Furthermore, resonances arising from the ACD monomer were attenuated to a greater extent than those of the ACD dimer in the presence of fibrils, suggesting that the monomer may bind fibrils. This hypothesis was supported by mutagenesis studies in which disulfide cross-linked ACD dimers formed by both αB-C and Hsp27 were less effective at inhibiting amyloid fibril elongation and fibril end-to-end joining than ACD constructs lacking disulfide cross-linking. Our results indicate that sHSP monomers inhibit amyloid fibril elongation, highlighting the importance of the dynamic oligomeric nature of sHSPs for client binding.

APOC3 Interference for Familial Chylomicronaemia Syndrome.

Patients with familial chylomicronaemia syndrome (FCS) have severe hypertriglyceridaemia due to genetically absent lipolytic capacity. They have a poor response to conventional therapies. To reduce the risk of potentially fatal pancreatitis, the management of FCS relies principally on a strict low-fat diet, which is difficult to follow and compromises quality of life. Targeted reduction of apolipoprotein C-III using new anti-APOC3 agents, such as the short interfering RNA ARO-APOC3, represents a promising approach to correct the severe biochemical disturbance in FCS.

N- and C-terminal regions of αB-crystallin and Hsp27 mediate inhibition of amyloid nucleation, fibril binding, and fibril disaggregation.

Small heat-shock proteins (sHSPs) are ubiquitously expressed molecular chaperones that inhibit amyloid fibril formation; however, their mechanisms of action remain poorly understood. sHSPs comprise a conserved α-crystallin domain flanked by variable N- and C-terminal regions. To investigate the functional contributions of these three regions, we compared the chaperone activities of various constructs of human αB-crystallin (HSPB5) and heat-shock 27-kDa protein (Hsp27, HSPB1) during amyloid formation by α-synuclein and apolipoprotein C-II. Using an array of approaches, including thioflavin T fluorescence assays and sedimentation analysis, we found that the N-terminal region of Hsp27 and the terminal regions of αB-crystallin are important for delaying amyloid fibril nucleation and for disaggregating mature apolipoprotein C-II fibrils. We further show that the terminal regions are required for stable fibril binding by both sHSPs and for mediating lateral fibril-fibril association, which sequesters preformed fibrils into large aggregates and is believed to have a cytoprotective function. We conclude that although the isolated α-crystallin domain retains some chaperone activity against amyloid formation, the flanking domains contribute additional and important chaperone activities, both in delaying amyloid formation and in mediating interactions of sHSPs with amyloid aggregates. Both these chaperone activities have significant implications for the pathogenesis and progression of diseases associated with amyloid deposition, such as Parkinson's and Alzheimer's diseases.

A Novel APOC2 Mutation in a Colombian Patient with Recurrent Hypertriglyceridemic Pancreatitis.

Hypertriglyceridemia is a common disease with only 2% of cases exhibiting monogenic mutations. Familial chylomicronemia syndrome (FCS) is a rare genetic condition associated with recurrent and severe episodes of pancreatitis and is mainly caused by mutations in the LPL gene, with few cases related to abnormal function of apolipoprotein C-II. This is a 50-year-old female with a past medical history of arterial hypertension, miscarriage and recurrent pancreatitis. In the last four years, her triglycerides and lipase concentration reached >3000 mg/dL and >700 U/L, respectively. The patient was not responsive to statins, fibrates, or tetrahydrolipstatin. A novel homozygous frameshift mutation on exon 3 of the APOC2 gene was detected, c.133_134delTC. Subsequent Sanger sequencing confirmed that three first-degree relatives were carriers of the same mutation. To the best of our knowledge, we are reporting the first Colombian patient with FCS due to an APOC2 mutation. We propose that this mutation caused recurrent hypertriglyceridemic pancreatitis.

Apolipoprotein C-II mimetic peptide is an efficient activator of lipoprotein lipase in human plasma as studied by a calorimetric approach.

Elevated plasma triglyceride (TG) levels are associated with higher risk of atherosclerotic cardiovascular disease. One way to reduce plasma TG is to increase the activity of lipoprotein lipase (LPL), the rate limiting enzyme in plasma TG metabolism. An apolipoprotein (apo) C-II mimetic peptide (18A-CII-a) has been recently developed that stimulated LPL activity in vitro and decreased plasma TG concentration in animal models for hypertriglyceridemia. Since this peptide can serve as a new therapeutic approach for treatment of hypertriglyceridemia, we investigated how 18A-CII-a peptide influences LPL activity in human plasma. We used recently described isothermal titration calorimetry based approach to assess the peptide, which enables the analysis in nearly undiluted human plasma. The 18A-CII-a peptide was 3.5-fold more efficient in stimulating LPL activity than full-length apoC-II in plasma sample from normolipidemic individual. Furthermore, 18A-CII-a also increased LPL activity in hypertriglyceridemic plasma samples. Unlike apoC-II, high concentrations of the 18A-CII-a peptide did not inhibit LPL activity. The increase in LPL activity after addition of 18A-CII-a or apoC-II to plasma was due to the increase of the amount of available substrate for LPL. Measurements with isolated lipoproteins revealed that the relative activation effects of 18A-CII-a and apoC-II on LPL activity were greater in smaller size lipoprotein fractions, such as remnant lipoproteins, low-density lipoproteins and high-density lipoproteins. In summary, this report describes a novel mechanism of action for stimulation of LPL activity by apoC-II mimetic peptides.

Lipid-apolipoprotein interactions in amyloid fibril formation and relevance to atherosclerosis.

The apolipoprotein family is a set of highly conserved proteins characterized by the presence of amphipathic α-helical sequences that mediate lipid binding. Paradoxically, this family of proteins is also prominent among the proteins known to form amyloid fibrils, characterized by extensive cross-ß structure. Several apolipoproteins including apolipoprotein (apo) A-I, apoA-II and apoC-II accumulate in amyloid deposits of atherosclerotic lesions. This review illustrates the role of lipid-apolipoprotein interactions in apolipoprotein folding and aggregation with a specific focus on human apoC-II, a well-studied member of the family. In the presence of high concentrations of micellar lipid mimetics apoC-II adopts a stable and predominantly α-helical structure, similar to other members of the family and presumed to be the structure of apoC-II in circulating plasma lipoproteins. In contrast, lipid-free apoC-II aggregates to form long amyloid fibrils with a twisted ribbon-like morphology. Detailed structural analyses identify a letter G-like conformation as the basic building block within these fibrils. Phospholipids at submicellar concentrations accelerate apoC-II fibril formation by promoting the formation of a discrete tetrameric intermediate. Conversely, several small molecule lipid-mimetics inhibit apoC-II fibril formation at submicellar concentrations, inducing well-defined dimers unable to further aggregate. Finally, low concentrations of phospholipid micelles and bilayers induce the slow formation of amyloid fibrils with distinct rod-like fibril morphology. These studies highlight the diversity of lipid effects on apolipoprotein amyloid formation and reveal a conformational adaptability that could underlie the widespread occurrence of apolipoproteins in amyloid deposits and atheroma.

Polymorphism in disease-related apolipoprotein C-II amyloid fibrils: a structural model for rod-like fibrils.

Human apolipoprotein (apo) C-II is one of several plasma apolipoproteins that form amyloid deposits in vivo and is an independent risk factor for cardiovascular disease. Lipid-free apoC-II readily self-assembles into twisted-ribbon amyloid fibrils but forms straight, rod-like amyloid fibrils in the presence of low concentrations of micellar phospholipids. Charge mutations exerted significantly different effects on rod-like fibril formation compared to their effects on twisted-ribbon fibril formation. For instance, the double mutant, K30D-D69K apoC-II, readily formed twisted-ribbon fibrils, while the rate of rod-like fibril formation in the presence of micellar phospholipid was negligible. Structural analysis of rod-like apoC-II fibrils, using hydrogen-deuterium exchange and NMR analysis showed exchange protection consistent with a core cross-ß structure comprising the C-terminal 58-76 region. Molecular dynamics simulations of fibril arrangements for this region favoured a parallel cross-ß structure. X-ray fibre diffraction data for aligned rod-like fibrils showed a major meridional spacing at 4.6 Å and equatorial spacings at 9.7, 23.8 and 46.6 Å. The latter two equatorial spacings are not observed for aligned twisted-ribbon fibrils and are predicted for a model involving two cross-ß fibrils in an off-set antiparallel structure with four apoC-II units per rise of the ß-sheet. This model is consistent with the mutational effects on rod-like apoC-II fibril formation. The lipid-dependent polymorphisms exhibited by apoC-II fibrils could determine the properties of apoC-II in renal amyloid deposits and their potential role in the development of cardiovascular disease.

[Severe hypertriglyceridemia : Diagnostics and new treatment principles]. / Schwere Hypertriglyzeridämie : Diagnostik und neue Therapieprinzipien.

Severe hypertriglyceridemia is defined at a plasma triglyceride (TG) concentration of >885 mg/dl and may result - in particular when clinical symptoms appear before the age of 40 - from "large variant" mutations in genes which influence the function of the lipoprotein lipase (LPL). For diagnosis, secondary factors have to be excluded and treated before further genetic tests are considered. Typical symptoms in almost all patients are recurrent, sometimes severe abdominal pain attacks, which can result in acute pancreatitis, the most important, sometimes life-threatening complication. To minimize the risk of severe pancreatitis, the aim is to maintain the plasma TG concentration <1000 mg/dl. Other clinical manifestations which can occur and are reversible are eruptive xanthomas, lipemia retinalis, hepatosplenomegaly, dyspnea syndrome, and impaired neurocognitive function. The hyperviscosity syndrome caused by chylomicronemia is seen as the underlying reason for some of the symptoms. Patients with mild-to-moderate hypertriglyceridemia have an increased cardiovascular risk. To lower this is the primary treatment goal here. Treatment mainly consists of a life-long, strict fat- and carbohydrate-restricted diet and the abstention from alcohol. Omega­3-Fatty acids and fibrates can be used to lower plasma TG levels. Recently, new gene therapy approaches for LPL-deficient patients have become available in Germany.

Novel Type of Renal Amyloidosis Derived from Apolipoprotein-CII.

Amyloidosis is characterized by extracellular deposition of misfolded proteins as insoluble fibrils. Most renal amyloidosis cases are Ig light chain, AA, or leukocyte chemotactic factor 2 amyloidosis, but rare hereditary forms can also involve the kidneys. Here, we describe the case of a 61-year-old woman who presented with nephrotic syndrome and renal impairment. Examination of the renal biopsy specimen revealed amyloidosis with predominant involvement of glomeruli and medullary interstitium. Proteomic analysis of Congo red-positive deposits detected large amounts of the Apo-CII protein. DNA sequencing of the APOC2 gene in the patient and one of her children detected a heterozygous c.206A→T transition, causing an E69V missense mutation. We also detected the mutant peptide in the proband's renal amyloid deposits. Using proteomics, we identified seven additional elderly patients with Apo-CII-rich amyloid deposits, all of whom had kidney involvement and histologically exhibited nodular glomerular involvement. Although prior in vitro studies have shown that Apo-CII can form amyloid fibrils and that certain mutations in this protein promote amyloid fibrillogenesis, there are no reports of this type of amyloidosis in humans. We propose that this study reveals a new form of hereditary amyloidosis (AApoCII) that is derived from the Apo-CII protein and appears to manifest in the elderly and preferentially affect the kidneys.

Triglyceride-rich lipoprotein metabolism in women: roles of apoC-II and apoC-III.

BACKGROUND: Experimental data suggest that apolipoprotein (apo) C-II and C-III regulate triglyceride-rich lipoprotein (TRL) metabolism, but there are limited studies in humans. We investigated the metabolic associations of TRLs with apoC-II and apoC-III concentrations and kinetics in women. MATERIAL AND METHODS: The kinetics of plasma apoC-II, apoC-III and very low-density lipoprotein (VLDL) apoB-100 and triglycerides were measured in the postabsorptive state using stable isotopic techniques and compartmental modelling in 60 women with wide-ranging body mass index (19·5-32·9 kg/m(2) ). RESULTS: Plasma apoC-II and apoC-III concentrations were positively associated with the concentrations of plasma triglycerides, VLDL1 - and VLDL2 -apoB-100 and triglyceride (all P < 0·05). ApoC-II production rate (PR) was positively associated with VLDL1 -apoB-100 concentration, VLDL1 triglyceride concentration and VLDL1 triglyceride PR, while apoC-II fractional catabolic rate (FCR) was positively associated with VLDL1 triglyceride FCR (all P < 0·05). No significant associations were observed between apoC-II and VLDL2 apoB-100 or triglyceride kinetics. ApoC-III PR, but not FCR, was positively associated with VLDL1 triglyceride, and VLDL2 -apoB-100 and triglyceride concentrations (all P < 0·05). No significant associations were observed between apoC-III and VLDL-apoB-100 and triglyceride kinetics. In multivariable analysis, including homoeostasis model assessment score, menopausal status and obesity, apoC-II concentration was significantly associated with plasma triglyceride, VLDL1 -apoB-100 and VLDL1 triglyceride concentrations and PR. Using the same multivariable analysis, apoC-III was significantly associated with plasma triglyceride and VLDL1 - and VLDL2 -apoB-100 and triglyceride concentrations and FCR. CONCLUSIONS: In women, plasma apoC-II and apoC-III concentrations are regulated by their respective PR and are significant, independent determinants of the kinetics and plasma concentrations of TRLs.

Apoc2 loss-of-function zebrafish mutant as a genetic model of hyperlipidemia.

Apolipoprotein C-II (APOC2) is an obligatory activator of lipoprotein lipase. Human patients with APOC2 deficiency display severe hypertriglyceridemia while consuming a normal diet, often manifesting xanthomas, lipemia retinalis and pancreatitis. Hypertriglyceridemia is also an important risk factor for development of cardiovascular disease. Animal models to study hypertriglyceridemia are limited, with no Apoc2-knockout mouse reported. To develop a genetic model of hypertriglyceridemia, we generated an apoc2 mutant zebrafish characterized by the loss of Apoc2 function. apoc2 mutants show decreased plasma lipase activity and display chylomicronemia and severe hypertriglyceridemia, which closely resemble the phenotype observed in human patients with APOC2 deficiency. The hypertriglyceridemia in apoc2 mutants is rescued by injection of plasma from wild-type zebrafish or by injection of a human APOC2 mimetic peptide. Consistent with a previous report of a transient apoc2 knockdown, apoc2 mutant larvae have a minor delay in yolk consumption and angiogenesis. Furthermore, apoc2 mutants fed a normal diet accumulate lipid and lipid-laden macrophages in the vasculature, which resemble early events in the development of human atherosclerotic lesions. In addition, apoc2 mutant embryos show ectopic overgrowth of pancreas. Taken together, our data suggest that the apoc2 mutant zebrafish is a robust and versatile animal model to study hypertriglyceridemia and the mechanisms involved in the pathogenesis of associated human diseases.

Apolipoprotein C-II Tuzla: a novel large deletion in APOC2 caused by Alu-Alu homologous recombination in an infant with apolipoprotein C-II deficiency.

BACKGROUNDS: Familial apolipoprotein (apo) C-II deficiency is a very rare inherited disorder characterized by chylomicronemia. Since the discovery in 1978, reports on apo C-II deficient patients have been limited and only 13 different mutations in APOC2, a gene encoding apo C-II protein, were identified. OBJECTIVES: The objective is to investigate the biochemical and genetic features of a 3-month-old Bosniak girl with chylomicronemia whose apo C-II protein was undetectable in her plasma. METHODS: APOC2, LPL, APOA5, and GPIHBP1 were sequenced. Isoelectrofocusing and immunoblotting of chylomicrons and VLDL fraction from the patient were performed. RESULTS: Sequence analysis demonstrated a large deletion of 2978 base pairs in APOC2, which encompassed exons 2, 3, and 4. The patient was homozygous for the deletion. The 5' part of the breakpoint was located in an Alu Sx repetitive element in intron 1 of APOC2, whereas the 3' part of the breakpoint was in another Alu Sx between APOC2 and CLPTM1, a gene flanking APOC2. We speculate that the deletion was caused by a homologous recombination between two Alu Sx elements. No mutations were detected in LPL, APOA5, and GPIHBP1. Isoelectrofocusing and immunoblotting confirmed the absence of apo C-II protein. CONCLUSIONS: We diagnosed the patient as having apo C-II deficiency and designated the novel large deletion as apo C-II Tuzla. This is the first description of apo C-II deficiency caused by Alu-Alu recombination in APOC2.