Plasma Proteomics and Lipid Profiles Analysis in Patients Affected by Coronary Artery Disease: Focus on Apolipoprotein C-III

Background: In the last decade Apolipoprotein C-III (Apo C-III) has been demonstrated to be a prognostic marker for cardiovascular risk on the basis of the strong correlation between Apo C-III plasma levels and high serum concentrations of triglyceride in humans. In such perspective, the molecular structure of Apo C-III may be relevant for its role Overall, very few studies tried to elucidate the impact of the Apo C-III concentration and modifications (sialylation) on lipoproteins and lipid metabolism and how Apo C-III can affect the outcome associated to Coronary Artery Disease (CAD) parameters. Methods: Three different groups of CAD patients, carefully selected among subjects enrolled in the Verona Heart Study project, were studied by means of proteomics and lipidomics technologies. Isoelectrofocusing and shotgun topdown MS approach were applied for the identification and quantification of the three different Apo C-III glycoforms. Mono and bidimensional western immunoblotting were performed in order to validate protein previously found by comparative analysis differentially expressed according to Apo CIII levels. A total proteomic profile of CAD patients was obtained by SWATH, an untargeted approach, analysis. Gas-Chromatography and Liquid-Chromatography-MS analysis allowed a lipidomic characterization of CAD and CAD free patients. Results: The three Apo C-III glycoforms showed a peculiar trend, where the non-sialylated form presented a negative correlation with the others parameters, instead the monosialylated a positive one. No correlations with the disialylated glycoforms were found. The validation analysis confirmed the comparative analysis results. The SWATH analysis underlined a peculiar set of proteins associated with low and high Apo C-III levels. The lipidomic approach underlined how according to Apo E levels in CAD and CAD free patients it is possible to observe a peculiar lipid profile, in particular high levels of Apo E are associated with the presence of cholesteryl ester oxidized. Conclusion: In spite of the relatively small sample size, the study allowed a multifaceted characterization of Apo C-III and Apo C-III glycoforms distribution in CAD patients. SWATH analysis revealed a set of proteins characterizing patients with high and low levels of Apo C-III. The lipidomic approach underlined that not only the apolipoproteins distribution and common lipids parameters but also some peculiar lipid species may play an important role in CAD. To validate the present results, further analysis are however required, possibly on larger populations of patients

Plasma proteomic analysis of patients with Coronary Artery Disease with different levels of apolipoprotein CIII

BACKGROUND: Coronary artery disease (CAD) is a multifactorial condition, involving alsogenetic factors. Among candidate genes associated with CAD risk there are lipoprotein lipase (LPL)gene and APOC3 gene. APOC3 encodes for the apolipoprotein CIII (ApoCIII) which acts as aninhibitor of LPL. Three different ApoCIII glycoforms (with different LPL inhibitory activity) -characterized by none, one or two sialic acids - have been described. Changes in the relativeabundance of these glycoforms have been observed in a variety of pathologies.The aim of this study was to analyze and quantify the apoCIII glycoforms and to assess theirrelationship with LPL activity and with the levels of the LPL activator apoA-V.METHODS: ApoCIII glycoforms in four groups of patients (from \u201cVerona Heart Study\u201d biobank,)classified according to the total plasma concentration of ApoCIII and different TG levels, wereanalyzed by a classical (isoelectric focusing/western blotting) and by a shotgun MS approach. LPLactivity (Fluorescent assay) and ApoA-V concentration (ELISA assay) were determined, and theircorrelations with lipid metabolism parameters were analyzed. We also validated by 2D-WB someproteins previously identified by 2-DE analysis.RESULTS Our results indicate that the distribution of the three ApoCIII glycoforms in the selectedgroups of patients are related to the TG levels, particularly the mono-sialylated isoform (ApoCIII-1)prevails in patients with the highest TG levels. A positive correlation between apoCIII and TGlevels as well a positive correlation between TG and more glycosylated ApoCIII were observed.Moreover, investigating the relation among ApoA-V level, LPL activity and the other parameters oflipid metabolism, we obtained useful information on their interplay as anti-atherogenic factor. Finally2D-WB patterns of serum proteins (i.e. Fibrinogen beta/gamma chain, Complement C3) confirmed thetrend in previously proteomics experiments.CONCLUSIONS: These data could provide important insights regarding the interaction amongapoCIII glycoforms, ApoA-V level and LPL activity in predicting cardiovascular risk inpredisposed individuals

ApoCIII glycoforms determination and proteomic analysis in plasma of coronary patients with different ApoCIII levels .

BACKGROUND:The aims of this study were:i) to analyze and quantify the apolipoproteinCIIIglycoforms characterized by none, one or two sialic acids,(having different lipoprotein lipase-LPLinhibitoryactivity), ii) to assess their relationship with LPL activity and ApoA-V in CAD patients,iii) to analyze some previously identified plasma proteins in relation to lipids status.METHODS: ApoCIII glycoforms in four groups of patients (from \u201cVerona Heart Study\u201d biobank,)classified according to the total plasma concentration of ApoCIII and different triglyceride (TG)levels, were analyzed by a classical (isoelectric focusing/western blotting) and by a shotgun MSapproach. LPL activity (Fluorescent assay) and ApoA-V concentration (ELISA assay) weredetermined, and their correlations with lipid metabolism parameters were analyzed. We alsovalidated by 2D-WB some plasma proteins previously identified by 2-DE analysis.RESULTS The distribution of the ApoCIII glycoforms in the selected groups of patients are relatedto the TG levels, particularly the mono-sialylated isoform (ApoCIII-1) prevails in patients with thehighest TG levels. Moreover, investigating the relation among ApoA-V level, LPL activity and theother parameters of lipid metabolism, we obtained useful information on their interplay as antiatherogenicfactors. ApoA-V concentration was found within the normal range, however higherthan previously reported. LPL activity measured as Vmax did not show significant correlations withApoCIII . Finally 2D-WB showed peculiar proteomic patterns of several proteins associated withelevated apoCIII plasma concentrations .CONCLUSIONS: As compared with the other ones, mono \u2013 sialylated isoform of apo CIII ispreferentially associated with TG levels. Samples with elevated levels of apoCIII are characterizedby specific proteomic patterns

Apolipoprotein -CIII glycoforms correlate heterogeneously with plasma lipid profile in subjects with coronary artery disease

AIMS: Apolipoprotein C-III (ApoC-III) is well recognized as a main determinant of triglyceride (TG) plasma concentration and plays a crucial role in coronary artery disease (CAD). However, data on ApoC-III glycoforms are only sparse so far. The aim of this study was to quantify ApoC-III glycoforms in CAD patients and to assess their correlations with plasma lipids. METHODS: ApoC-III glycoforms were analysed by mass spectrometry in 55 subjects with clinically stable CAD (90.9% males, mean age 70.2\ub17.9 years) within the framework of the Verona Heart Study. RESULTS: Three different ApoC-III glycoforms were identified: non-sialylated (ApoC-III0), monosialylated (ApoC-III1), and disialylated isoforms (ApoC-III2). The analyses were performed on the relative proportion of ApoC-III glycoforms. ApoC-III0 correlated negatively with total ApoC-III concentration (R=-0.351;P=0.009), ApoC-III1 had a positive correlation (R=0.382;P=0.004), while ApoC-III2 did not have significant correlation. The different glycoforms showed different pattern of correlations with the other parameters of plasma lipid profile. ApoC-III0 was inversely correlated with TG (R=-0.421;P=0.001), total cholesterol (R=-0.314;P=0.020), LDL cholesterol (R=-0.317;P=0.018), ApoE (R=-0.434;P=0.001), and Apo B (R=-0.292;P=0.030), while ApoC-III1 was directly correlated with TG (R=0.438;P=0.001) ), total cholesterol (R=0.280;P=0.038), LDL cholesterol (R=0.337;P=0.012), ApoE (R=0.457;P=0.001), and ApoB (R=0.288;P=0.033). On the other hand, no significant correlation was found for ApoC-III2. The ApoC-III1/ApoC-III0 ratio was a significant predictor of both TG (\u3b2=0.201;P=0.025) and ApoE levels (\u3b2=0.270; P=0.016) in linear regression models adjusted for sex, age, and total ApoC-III concentration.CONCLUSIONS: ApoC-III glycoforms have heterogeneous correlations with plasma lipids in CAD patients, suggesting that they could have different metabolic properties and biological activities

Increased urinary excretion of the epithelial Na channel activator prostasin in patients with primary aldosteronism

OBJECTIVES: Prostasin is a glycosylphosphatidylinositol-anchored serine protease that is released in urine and is involved in epithelial Na channel activation. A direct association between urinary prostasin (u-prostasin) concentration and activation of the aldosterone-driven pathway has been suggested; however, in previous studies on primary aldosteronism, a semiquantitative evaluation, rather than a precise quantification, of prostasin was performed. We aim to investigate if u-prostasin concentrations are higher in patients with primary aldosteronism than in patients with essential hypertension and whether u-prostasin measurements could be a useful marker for diagnosing primary aldosteronism in hypertensive patients. METHODS: A total of 62 primary aldosteronism and 56 essential hypertension patients were enrolled. Biochemical and hormonal parameters were measured by applying routine laboratory methods, and u-prostasin levels were assessed by ELISA. RESULTS: Primary aldosteronism patients had higher u-prostasin levels than did essential hypertension patients. Prostasin levels were positively correlated with the aldosterone-to-renin ratio and inversely correlated with plasma K and urinary Na levels. In the highest concentration quartile, u-prostasin levels were associated with a several-fold higher probability of primary aldosteronism diagnosis in hypertensive patients. Receiver operating characteristic curve analysis showed that prostasin was specific but poorly sensitive as a diagnostic marker for primary aldosteronism. CONCLUSIONS: The study shows that an elevated u-prostasin concentration in humans is a specific marker for primary aldosteronism, which involves the classical model of epithelial Na channel activation. There was no statistically significant difference in prostasin concentrations among patients with different primary aldosteronism subtypes. Studies with a larger series of patients are necessary to clarify the clinical usefulness of the prostasin assay

Ceruloplasmin impairs endothelium-dependent relaxation of rabbit aorta.

This study evaluated the effects of ceruloplasmin, the copper-containing blue oxidase of vertebrate plasma, on the relaxation of rabbit aortic rings after endothelial release of nitric oxide (NO). Ceruloplasmin at physiological, i.e., micromolar, concentrations inhibited relaxation of rabbit aorta induced by endothelium-dependent agonists hire acetylcholine or ADP, whereas it was ineffective toward vasodilation due to direct stimulation of smooth muscle cells by nitroglycerin. The effect was reversible and specific for native, fully metalated ceruloplasmin, since relaxation was not impaired by the heat-treated or metal-depleted derivatives. A trapping mechanism, involving a direct interaction of NO or other NO-containing species (like nitrosothiols and iron-dinitrosyls) with the copper sites and/or with the free thiol of ceruloplasmin, could be safely excluded on the basis of spectroscopic and chemical analyses of the protein exposed to authentic NO, nitrosothiols, or iron-dinitrosyls. The data presented in this paper constitute the first evidence of impairment of the endothelium-dependent vasodilatation by a plasma protein and may shed some Light on the still uncertain physiological role of ceruloplasmin

Plasma Proteome Profiles of Stable CAD Patients Stratified According to Total Apo C-III Levels

The present research reports the study the of plasma proteome profile of stable coronary artery disease (CAD) patients characterized by different levels of total Apolipoprotein-CIII (Apo C-III), a prognostic marker for cardiovascular risk

Sialylated isoforms of apolipoprotein C-III and plasma lipids in subjects with coronary artery disease

Background: Apolipoprotein C-III (ApoC-III), a key regulator of plasma triglyceride (TG), is present in three isoforms, i.e. non-sialylated (ApoC-III0), monosialylated (ApoC-III1) and disialylated (ApoC-III2). We aimed at quantifying the distribution of the ApoC-III glycoforms in patients with angiographically demonstrated coronary artery disease (CAD) according to levels of total ApoC-III plasma concentration.Methods: ApoC-III glycoforms were quantified by a specifically developed, high-resolution, mass spectrometry method in unrelated CAD patients. Lipoprotein lipase (LPL) activity was estimated by a fluorescence-based method.Results: In 101 statin-treated CAD patients, the absolute concentrations of the three glycoforms similarly increased across ApoC-III quartiles, but the proportion of ApoC-III1 rose whereas that of ApoC-III0 decreased progressively by increasing total ApoC-III concentrations. The proportion of ApoC-III2 was quite constant throughout the whole range of total ApoC-III. A higher proportion of ApoC-III1 reflected an unfavorable lipid profile characterized by high levels of TG, total and low density lipoprotein cholesterol, ApoE and reduced ApoA-I. The correlations between ApoC-III glycoforms and TG were confirmed in 50 statin-free CAD patients. High concentration of total ApoC-III was associated with low LPL activity, while no correlation was found for the relative proportion of glycoforms.Conclusions: Specific patterns of ApoC-III glycoforms are present across different total ApoC-III concentrations in CAD patients. The inhibitory effect of ApoC-III on LPL appears related to total ApoC-III concentration, but not to the relative proportion of ApoC-III glycoforms