ApoC-III is a novel inducer of calcification in human aortic valves.

Calcific aortic valve disease (CAVD) occurs when subpopulations of valve cells undergo specific differentiation pathways, promoting tissue fibrosis and calcification. Lipoprotein particles carry oxidized lipids that promote valvular disease, but low-density lipoprotein-lowering therapies have failed in clinical trials, and there are currently no pharmacological interventions available for this disease. Apolipoproteins are known promoters of atherosclerosis, but whether they possess pathogenic properties in CAVD is less clear. To search for a possible link, we assessed 12 apolipoproteins in nonfibrotic/noncalcific and fibrotic/calcific aortic valve tissues by proteomics and immunohistochemistry to understand if they were enriched in calcified areas. Eight apolipoproteins (apoA-I, apoA-II, apoA-IV, apoB, apoC-III, apoD, apoL-I, and apoM) were enriched in the calcific versus nonfibrotic/noncalcific tissues. Apo(a), apoB, apoC-III, apoE, and apoJ localized within the disease-prone fibrosa and colocalized with calcific regions as detected by immunohistochemistry. Circulating apoC-III on lipoprotein(a) is a potential biomarker of aortic stenosis incidence and progression, but whether apoC-III also induces aortic valve calcification is unknown. We found that apoC-III was increased in fibrotic and calcific tissues and observed within the calcification-prone fibrosa layer as well as around calcification. In addition, we showed that apoC-III induced calcification in primary human valvular cell cultures via a mitochondrial dysfunction/inflammation-mediated pathway. This study provides a first assessment of a broad array of apolipoproteins in CAVD tissues, demonstrates that specific apolipoproteins associate with valvular calcification, and implicates apoC-III as an active and modifiable driver of CAVD beyond its potential role as a biomarker.

ApoE and apoC-III-defined HDL subtypes: a descriptive study of their lecithin cholesterol acyl transferase and cholesteryl ester transfer protein content and activity.

BACKGROUND: The functionality of high-density lipoproteins (HDL) is a better cardiovascular risk predictor than HDL concentrations. One of the key elements of HDL functionality is its apolipoprotein composition. Lecithin-cholesterol acyl transferase (LCAT) and cholesterol-ester transfer protein (CETP) are enzymes involved in HDL-mediated reverse cholesterol transport. This study assessed the concentration and activity of LCAT and CETP in HDL subspecies defined by their content of apolipoproteins E (apoE) and C-III (apoC-III) in humans. METHODS: Eighteen adults (ten women and eight men, mean age 55.6, BMI 26.9 Kg/m2, HbA1c 5.4%) were studied. HDL from each participant were isolated and divided into four subspecies containing respectively: No apoE and no apoC-III (E-C-), apoE but not apoC-III (E + C-), apoC-III but no apoE (E-C+) and both apoE and apoC-III (E + C+). The concentration and enzymatic activity of LCAT and CETP were measured within each HDL subspecies using immunoenzymatic and fluorometric methods. Additionally, the size distribution of HDL in each apolipoprotein-defined fraction was determined using non-denaturing electrophoresis and anti-apoA-I western blotting. RESULTS: HDL without apoE or apoC-III was the predominant HDL subtype. The size distribution of HDL was very similar in all the four apolipoprotein-defined subtypes. LCAT was most abundant in E-C- HDL (3.58 mg/mL, 59.6% of plasma LCAT mass), while HDL with apoE or apoC-III had much less LCAT (19.8, 12.2 and 8.37% of plasma LCAT respectively for E + C-, E-C+ and E + C+). LCAT mass was lower in E + C- HDL relative to E-C- HDL, but LCAT activity was similar in both fractions, signaling a greater activity-to-mass ratio associated with the presence of apoE. Both CETP mass and CETP activity showed only slight variations across HDL subspecies. There was an inverse correlation between plasma LCAT activity and concentrations of both E-C+ pre-beta HDL (r = - 0.55, P = 0.017) and E-C- alpha 1 HDL (r = - 0.49, P = 0.041). Conversely, there was a direct correlation between plasma CETP activity and concentrations of E-C+ alpha 1 HDL (r = 0.52, P = 0.025). CONCLUSIONS: The presence of apoE in small HDL is correlated with increased LCAT activity and esterification of plasma cholesterol. These results favor an interpretation that LCAT and apoE interact to enhance anti-atherogenic pathways of HDL.

Complexity, cost, and content - three important factors for translation of clinical protein mass spectrometry tests, and the case for apolipoprotein C-III proteoform testing.

Complexity, cost, and content are three important factors that can impede translation of clinical protein mass spectrometry (MS) tests at a larger scale. Complexity stems from the many components/steps involved in bottom-up protein MS workflows, making them significantly more complicated than enzymatic immunoassays (EIA) that currently dominate clinical testing. This complexity inevitably leads to increased costs, which is detrimental in the price-competitive clinical marketplace. To successfully compete, new clinical protein MS tests need to offer something new and unique that EIAs cannot - a new content of proteoform detection. The preferred method for proteoform profiling is intact protein MS analysis, in which all proteins are measured as intact species thus allowing discovery of new proteoforms. To illustrate the importance of intact proteoform testing with MS and its potential clinical implications, we discuss here recent findings from multiple studies on the distribution of apolipoprotein C-III proteoforms and their correlations with key clinical measures of dyslipidemia. Such studies are only made possible with assays that are low in cost, avoid unnecessary complexity, and are unique in providing the content of proteoforms.

A capillary zone electrophoresis method for detection of Apolipoprotein C-III glycoforms and other related artifactually modified species.

ApolipoproteinC-III (ApoC-III) is a human plasma glycoprotein whose O-glycosylation can be altered as a result of congenital disorders of glycosylation (CDG). ApoC-III exhibits three major glycoforms whose relative quantification is of utmost importance for the diagnosis of CDG patients. Considering the very close structures of these glycoforms and their tendency to adsorb on the capillary, a thorough optimization of capillary electrophoresis (CE) parameters including preconditioning and in-between rinsing procedures was required to efficiently separate all the ApoC-III glycoforms. Permanent coatings did not contribute to high resolution separations. A fast and reliable method based on a bare-silica capillary combining the effect of urea and diamine additives allowed to separate up to six different ApoC-III forms. We demonstrated by a combination of MALDI-TOF mass spectrometry (MS) analyses and CE of intact and neuraminidase-treated samples that this method well resolved glycoforms differing not only by their sialylation degree but also by carbamylation state, an undesired chemical modification of primary amines. This method allowed to demonstrate the carbamylation of ApoC-III glycoforms for the first time. Our CZE method proved robust and accurate with excellent intermediate precision regarding migration times (RSDs < 0.7%) while RSDs for peak areas were less than 5%. Finally, the quality of three distinct batches of commercial ApoC-III obtained from different suppliers was assessed and compared. Quite similar but highly structurally heterogeneous ApoC-III profiles were observed for these samples.

Characterization of Apolipoprotein C3 (Apo C3) LNA/DNA Impurities and Degradation Products by LC-MS/MS.

Apolipoprotein C3 (Apo C3) LNA/DNA gapmer was evaluated under various stress and formulation conditions for the purpose of its development as a potential biotherapeutic for low density lipoprotein (LDL) lowering. Using ion-pairing (IP) reversed-phase (RP) liquid chromatography ultra-high resolution (UHR) tandem mass spectrometry (IP-RPLC-MS/MS), a combination of accurate mass measurements and collision-induced dissociation enabled in-depth characterization of Apo C3 LNA/DNA oligonucleotide, in particular the inherent impurities following synthesis and degradation products after exposure to stress conditions. In this study, oligonucleotide samples were stressed under different pH and UV exposure conditions. The primary impurities in Apo C3 LNA/DNA were losses of nucleotide moieties from both the 5'- and 3'-terminus leading to n-1, n-2, etc. species. Desulfurization and depurination were observed in Apo C3 LNA/DNA after a week under UV light stress conditions at low pH. Guanine oxidation and dimerization were the primary degradation products detected under UV light exposure for 1 week at high pH. The effect of antioxidants on the levels of these degradation products was evaluated under neutral pH conditions. In the presence of all antioxidants, levels of guanine oxidation and desulfurization under tested conditions were the same as those in the unstressed sample, except for sodium ascorbate. The thorough understanding of the Apo C3 LNA/DNA oligonucleotide structure, its impurities, and degradation products laid the foundation for the successful formulation development of this novel biotherapeutic modality.

Mass spectrometric immunoassays for discovery, screening and quantification of clinically relevant proteoforms.

Human proteins can exist as multiple proteoforms with potential diagnostic or prognostic significance. MS top-down approaches are ideally suited for proteoforms identification because there is no prerequisite for a priori knowledge of the specific proteoform. One such top-down approach, termed mass spectrometric immunoassay utilizes antibody-derivatized microcolumns for rapid and contained proteoforms isolation and detection via MALDI-TOF MS. The mass spectrometric immunoassay can also provide quantitative measurement of the proteoforms through inclusion of an internal reference standard into the analytical sample, serving as normalizer for all sample processing and data acquisition steps. Reviewed here are recent developments and results from the application of mass spectrometric immunoassays for discovery of clinical correlations of specific proteoforms for the protein biomarkers RANTES, retinol binding protein, serum amyloid A and apolipoprotein C-III.

Contribución de la apolipoproteína CIII a la aterogenicidad de las dislipidemias / Apolipoprotein CIII contribution to atherogenicity of dyslipidemias

La apolipoproteína CIII (apoCIII) inhibe la hidrólisis de triglicéridos de la VLDL y quilomicrones por la lipasa lipoproteica. Se ha pensado que la hipertrigliceridemia resultante explica la asociación entre el aumento de la apoCIII con exageración del riesgo cardiovascular. Sin embargo, estudios clínicos indican que un aumento de la apoCIII asociada a las LDL es un factor de riesgo independiente de la hipertrigliceridemia. Experimentos recientes indican que la apoCIII incrementa la adhesión de monocitos al endotelio, un requisito para la acumulación de macrófagos en la aterogénesis. Además, LDL ricas en apoCIII tienen una alta afinidad por los proteoglicanos de la íntima, lo cual podría aumentar su deposición subendotelial. Estas dos propiedades y efectos de la LDL rica en apoCIII pueden ser la base de los hallazgos indicativos de que la apoCIII es un agente causal de la aterogénesis y un factor de riesgo cardiovascular independiente (AU)

Summary The apolipoprotein CIII (apoCIII) is an inhibitor of the hydrolysis of triglycerides inchylomicrons and VLDL by lipoprotein lipase. It has been accepted that the hypertriglyceridemia induced by high apoCIII can explain its association with increased cardiovascular risk.However, several studies indicate that increased apoCIII is a risk factor independent of hypertriglyceridemia. Recent experimental evidence in vitro and in vivo indicates that apoCIII byitself, or associated with lipoproteins, increase expression of cell adhesion molecules in theendothelium and monocytes. Thus causing associations between these cells. In addition, it hasbeen shown that LDL rich in apoCIII have a high affinity for proteoglycans of the arterial intima.A property that could increase the rate of LDL entrapment in the subendothelial intima. Thesetwo properties of LDL rich in apoCIII indicate that apoCIII is a significant risk factor because itcan have a causal role in atherogenesis (AU)

Quantitative analysis of intact apolipoproteins in human HDL by top-down differential mass spectrometry.

Top-down mass spectrometry holds tremendous potential for the characterization and quantification of intact proteins, including individual protein isoforms and specific posttranslationally modified forms. This technique does not require antibody reagents and thus offers a rapid path for assay development with increased specificity based on the amino acid sequence. Top-down MS is efficient whereby intact protein mass measurement, purification by mass separation, dissociation, and measurement of product ions with ppm mass accuracy occurs on the seconds to minutes time scale. Moreover, as the analysis is based on the accurate measurement of an intact protein, top-down mass spectrometry opens a research paradigm to perform quantitative analysis of "unknown" proteins that differ in accurate mass. As a proof of concept, we have applied differential mass spectrometry (dMS) to the top-down analysis of apolipoproteins isolated from human HDL(3). The protein species at 9415.45 Da demonstrates an average fold change of 4.7 (p-value 0.017) and was identified as an O-glycosylated form of apolipoprotein C-III [NANA-(2 --> 3)-Gal-beta(1 --> 3)-GalNAc, +656.2037 Da], a protein associated with coronary artery disease. This work demonstrates the utility of top-down dMS for quantitative analysis of intact protein mixtures and holds potential for facilitating a better understanding of HDL biology and complex biological systems at the protein level.

Mass spectrometric identification of aberrantly glycosylated human apolipoprotein C-III peptides in urine from Schistosoma mansoni-infected individuals.

Schistosomiasis is a parasitic infection caused by Schistosoma flatworms, prime examples of multicellular parasites that live in the mammalian host for many years. Glycoconjugates derived from the parasite have been shown to play an important role in many aspects of schistosomiasis, and some of them are present in the circulation of the host. The aim of this study was to identify novel glycoconjugates related to schistosomiasis in urine of Schistosoma mansoni-infected individuals using a combination of glycopeptide separation techniques and in-depth mass spectrometric analysis. Surprisingly, we characterized a heterogeneous population of novel aberrantly O-glycosylated peptides derived from the C terminus of human apolipoprotein C-III (apoC-III) in urine of S. mansoni-infected individuals that were not detected in urine of non-infected controls. The glycan composition of these glycopeptides is completely different from what has been described previously for apoC-III. Most importantly, they lack sialylation and display a high degree of fucosylation. This study exemplifies the potential of mass spectrometry for the identification and characterization of O-glycopeptides without prior knowledge of either the glycan or the peptide sequence. Furthermore, our results indicate for the first time that as a result of S. mansoni infection the glycosylation of a host protein is altered.

Effect of weight loss on markers of triglyceride-rich lipoprotein metabolism in the metabolic syndrome.

BACKGROUND: Hypertriglyceridaemia, a consistent feature of dyslipidaemia in the metabolic syndrome (MetS), is related to the extent of abdominal fat mass and altered adipocytokine secretion. We determined the effect of weight loss by dietary restriction on markers of triglyceride-rich lipoprotein (TRL) metabolism and plasma adipocytokines. DESIGN: Thirty-five men with MetS participated in a 16 week randomized controlled dietary intervention study. Apolipoprotein (apo) C-III, apoB-48, remnant-like particle (RLP)-cholesterol, total adiponectin, high-molecular weight (HMW) adiponectin, and retinol-binding protein-4 (RBP-4) concentrations were measured using immunoassays. RESULTS: Compared with weight maintenance (n = 15), weight loss (n = 20) significantly decreased body weight, plasma insulin, triglycerides, total cholesterol, low-density lipoprotein (LDL)-cholesterol and lathosterol (P < 0.05). Weight loss also decreased plasma concentrations of apoC-III (-33%), apoB-48 (-37%), very low-density lipoprotein (VLDL)-apoB (-43%), RLP-cholesterol (-48%), and RBP-4 (-20%), and significantly increased plasma total (+20%) and HMW-adiponectin (+19%) concentrations. In the weight loss group, reduction in plasma apoC-III was associated (P < 0.05) with reduction in plasma apoB-48, VLDL-apoB, RLP-cholesterol and triglycerides. Increase in total adiponectin was associated (P < 0.05) with the reduction in plasma VLDL-apoB and triglycerides. The changes in HMW-adiponectin and RBP-4 were not associated with changes in plasma apoB-48, apoC-III, VLDL-apoB, RLP-cholesterol or triglycerides. In multiple regression analysis including changes in visceral fat, insulin and total adiponectin concentrations, the fall in plasma apoC-III concentration was an independent predictor of the reductions in plasma apoB-48, VLDL-apoB, RLP-cholesterol and triglycerides concentrations. CONCLUSIONS: In men with MetS, weight loss decreases the plasma concentrations of apoB-48, VLDL-apoB, RLP-cholesterol and triglycerides. This effect could partly relate to concomitant changes in plasma apoC-III and adiponectin concentrations that accelerate the catabolism of TRLs.

The function, composition, and particle size of high-density lipoprotein were severely impaired in an oliguric phase of hemorrhagic fever with renal syndrome patients.

BACKGROUND: Patients suffering from hemorrhagic fever with renal syndrome (HFRS) often showed strikingly reduced high-density lipoprotein (HDL)-cholesterol levels during the oliguric phase, indicating severe alterations in lipoprotein metabolism. OBJECTIVE: To compare changes in the functions and composition of HDL, lipoprotein metabolism parameters were analyzed in the sera of HFRS patients in the oliguric phase and after recovery. METHODS: The serum cholesterol, triglyceride (TG), and lipoprotein/apolipoprotein profiles of HFRS patients in the oliguric and recovery phases were compared with those of normal reference sera. The activities of HDL-associated enzymes, lecithin:cholesterol acyltransferase (LCAT), and paraoxonase (PON) were also assessed. RESULTS: In the oliguric phase, serum cholesterol was substantially decreased and serum TG was increased. As observed by electron microscopy, the sizes of the HDL particles from the HFRS patients were smaller than those seen in the reference sera, with more heterogeneous distribution. Serum amyloid A (SAA) and apolipoprotein (apo) C-III were overexpressed in the oliguric phase, particularly in the HDL fraction. However, in immunodetection, the levels of apoA-I in the HDL(2) and HDL(3) of the HFRS patients were lower than those of the reference HDL. Serum LCAT and PON activities were reduced significantly in the oliguric phase, which is associated with a reduction in HDL-cholesterol levels and HDL particle size. CONCLUSION: Overexpression of both apoC-III and apoSAA in HDL and attenuated serum LCAT and PON activity were observed during the oliguric phase in HFRS patients. These results demonstrate that structural, functional, and compositional changes of HDL occurred to a substantial degree in the oliguric phase.

Dynamics of dense electronegative low density lipoproteins and their preferential association with lipoprotein phospholipase A(2).

Small, dense, electronegative low density lipoprotein [LDL(-)] is increased in patients with familial hypercholesterolemia and diabetes, populations at increased risk for coronary artery disease. It is present to a lesser extent in normolipidemic subjects. The mechanistic link between small, dense LDL(-) and atherogenesis is not known. To begin to address this, we studied the composition and dynamics of small, dense LDL(-) from normolipidemic subjects. NEFA levels, which correlate with triglyceride content, are quantitatively linked to LDL electronegativity. Oxidized LDL is not specific to small, dense LDL(-) or lipoprotein [a] (i.e., abnormal lipoprotein). Apolipoprotein C-III is excluded from the most abundant LDL (i.e., that of intermediate density: 1.034 < d < 1.050 g/ml) but associated with both small and large LDL(-). In contrast, lipoprotein-associated phospholipase A(2) (LpPLA(2)) is highly enriched only in small, dense LDL(-). The association of LpPLA(2) with LDL may occur through amphipathic helical domains that are displaced from the LDL surface by contraction of the neutral lipid core.