Impact of PCSK9 inhibitors in glycaemic control and new-onset diabetes.

BACKGROUND: The diabetogenic effect of statins has been well established by clinical trials, Mendelian randomisation studies and meta-analyses. According to large clinical trials, PCSK9 inhibitors (PCSK9i) have no deleterious impact on glucose metabolism. However, few real-life studies have yet evaluated the long-term effects of these drugs on glucose homeostasis and their impact on new-onset diabetes (NODM). METHODS: We studied 218 patients treated with either alirocumab or evolocumab (70% with familial hypercholesterolemia) for at least three years (PCSK9iG). We studied the NODM rate in the nondiabetic group at baseline (168) and overall glucose metabolism control in the whole group. Incidental DM was compared with two groups. The first was a propensity score matching (PSM)-selected group (n = 168) from the database of patients attending the Reus lipid unit (Metbank, n = 745) who were not on PCSK9i (PSMG). The second was a subgroup with a similar age range (n = 563) of the Di@bet.es study (Spanish prospective study on diabetes development n = 5072) (D@G). The incidence was reported as the percentage of NODM cases per year. RESULTS: The fasting glucose (FG) level of the subjects with normoglycaemia at baseline increased from 91 (86-95.5) to 93 (87-101) mg/dL (p = 0.014). There were 14 NODM cases in the PCSK9i group (2.6%/y), all among people with prediabetes at baseline. The incidence of NODM in PSMG and D@G was 1.8%/y (p = 0.69 compared with the PCSK9iG). The incidence among the subjects with prediabetes was 5.1%/y in the PCSK9iG, 4.8%/y in the PSMG and 3.9%/y in the D@G (p = 0.922 and p = 0.682, respectively). In the multivariate analysis, only the FG level was associated with the development of NODM in the PCSK9iG (OR 1.1; 95% CI: 1.0-1.3; p = 0.027). Neither FG nor A1c levels changed significantly in patients with DM at baseline. CONCLUSION: A nonsignificant increase in NODM occurred in the PCSK9iG, particularly in patients with prediabetes, compared with the PSMG and D@G groups. Baseline FG levels were the main variable associated with the development of DM. In the subjects who had DM at baseline, glucose control did not change. The impact of PCSK9i on glucose metabolism should not be of concern when prescribing these therapies.

Lipidomics of triglyceride-rich lipoproteins derived from hyperlipidemic patients on inflammation.

BACKGROUND AND AIM: Triglyceride-rich lipoproteins (TRLs) can have an important role in atherosclerosis development due to their size and ability to penetrate the endothelium. While high plasma triglyceride (TG) levels and chronic inflammation are relevant in metabolic diseases, it remains unclear whether TGs are atherogenic or which TRL-TG-derived metabolites are responsible for inflammation. Here, we aimed to study the lipidome modifications of TRL particles enriched in TG in patients with hyperlipidemia and their associations with a proinflammatory status both in vivo and in vitro. METHODS: Using proton nuclear magnetic resonance (1 H-NMR), we analysed the plasma levels of glycoprotein acetyls and the TRL lipidomic profile of 307 patients with dyslipidemia. THP-1-derived macrophages were used as an in vitro model to explore the molecular inflammatory effects mediated by TRL. RESULTS: In vivo, higher TRL-TG levels were associated with higher circulating levels of NMR-measured glycoproteins (Glyc-A, Glyc-B and Glyc-F; p < .001). Lipidomic analysis showed that TRL-TG enrichment led to decreased cholesterol and phospholipid content (p < .01), an increase in omega-9, and a decrease in saturated fatty acids (p < .001). THP-1 macrophages exposed to increasing TRL particle concentrations augmented the secretion of IL-1ß and TNF-α, which varied based on particle composition. Particles with higher cholesterol and phospholipid contents exerted higher cytokine secretion. The activation of MAPK, Akt/NFκB, and caspase-1 was concurrent with this proinflammatory response. CONCLUSIONS: High TRL-TG levels are associated with a higher systemic inflammatory status and increased particle concentrations. In vitro, higher particle numbers increase proinflammatory cytokine secretion, with cholesterol and phospholipid-rich TRL being more proinflammatory.

Lipidomics Reveals Myocardial Lipid Composition in a Murine Model of Insulin Resistance Induced by a High-Fat Diet.

Ectopic fat accumulation in non-adipose tissues is closely related to diabetes-related myocardial dysfunction. Nevertheless, the complete picture of the lipid metabolites involved in the metabolic-related myocardial alterations is not fully characterized. The aim of this study was to characterize the specific lipid profile in hearts in an animal model of obesity/insulin resistance induced by a high-fat diet (HFD). The cardiac lipidome profiles were assessed via liquid chromatography-mass spectrometry (LC-MS)/MS-MS and laser desorption/ionization-mass spectrometry (LDI-MS) tissue imaging in hearts from C57BL/6J mice fed with an HFD or standard-diet (STD) for 12 weeks. Targeted lipidome analysis identified a total of 63 lipids (i.e., 48 triacylglycerols (TG), 5 diacylglycerols (DG), 1 sphingomyelin (SM), 3 phosphatidylcholines (PC), 1 DihydroPC, and 5 carnitines) modified in hearts from HFD-fed mice compared to animals fed with STD. Whereas most of the TG were up-regulated in hearts from animals fed with an HFD, most of the carnitines were down-regulated, thereby suggesting a reduction in the mitochondrial ß-oxidation. Roughly 30% of the identified metabolites were oxidated, pointing to an increase in lipid peroxidation. Cardiac lipidome was associated with a specific biochemical profile and a specific liver TG pattern. Overall, our study reveals a specific cardiac lipid fingerprint associated with metabolic alterations induced by HFD.

A panel of plasma microRNAs improves the assessment of surrogate markers of cardiovascular disease in rheumatoid arthritis patients.

OBJECTIVE: Patients with RA present increased risk of cardiovascular (CV) disease compared with the general population. Moreover, CV risk factors that have a causal relationship with atherosclerosis do not seem to fully explain the accelerated process that they exhibit. We evaluated the association of a 10 microRNAs panel with surrogate markers of subclinical arteriosclerosis [carotid intima-media thickness (cIMT), carotid plaque presence (cPP), pulse wave velocity (PWV) and distensibility] in a cohort of RA patients. MATERIAL AND METHODS: A total of 199 patients with RA were included. Surrogate markers of arteriosclerosis were measured with My Lab 60 X-Vision sonographer. MicroRNAs were extracted from plasma and quantified with qPCR. Multivariate models and classification methods were performed. RESULTS: Multivariate models showed that microRNAs-24 (ß = 15.48), 125a (ß = 9.93), 132 (ß = 11.52), 146 (ß = 15.12), 191 (ß = 13.25) and 223 (ß = 13.30) were associated with cIMT globally. MicroRNA-24 [odds ratio (OR) = 0.41], 146 (OR = 0.36) and Let7a (OR = 0.23) were associated with cPP in men. Including the microRNAs in a partial least square discriminant analysis model properly classified men with and without cPP. MicroRNA-96 (ß = -0.28) was associated with PWV in male patients. Finally, several miRNAs were also associated with cIMT, cPP and arterial stiffness in the high DAS28 group and in the earlier tertile groups of disease duration. CONCLUSION: Plasmatic expression of microRNA-24, 96, 103, 125a, 132, 146, 191, 223 and Let7a were associated with surrogate markers of CV disease and could be predictors of CV risk in patients with RA.

Serum branch-chained amino acids are increased in type 2 diabetes and associated with atherosclerotic cardiovascular disease.

BACKGROUND AND AIM: Circulating biomarkers of metabolic and cardiovascular diseases can help in the early detection and prevention of those diseases. Using proton nuclear magnetic resonance (1H-NMR), we aimed to study the plasma levels of low-molecular-weight metabolites (LMWMs) in a cohort of 307 patients with metabolic diseases to assess their relationships with type-2 diabetes (T2D) and incident atherosclerotic cardiovascular disease (ASCVD). METHODS: We conducted a cross-sectional and prospective study. We included 307 patients attending the Lipid Unit of our University Hospital for the treatment of the following metabolic disturbances and associated disorders: T2D (73.9%), obesity (58.7%), and hypertension (55.1%). 1H-NMR was used to study the plasma levels of 13 LMWMs. LMWM serum concentrations were evaluated in patients with and without T2D. and the correlations with several parameters and their associations with T2D were analyzed. The association between LMWM levels at baseline and the development of ASCVD in patients with T2D after 10 years of follow-up was also evaluated. RESULTS: Among the LMWMs measured, the branched-chain amino acids (BCAAs) valine, leucine and isoleucine showed a positive association with several clinical and lipid-related biochemical parameters and inflammatory markers (p < 0.05). Likewise, these three BCAAS were associated with diabetes even after adjusting for covariates (p < 0.05). During the follow-up period of 10 years, 29 of the 185 patients with diabetes at baseline (15.68%) developed ASCVD. After adjusting for clinical covariates, baseline levels of valine and alanine were associated with the development of ASCVD (p < 0.05). CONCLUSION: Overall, our results indicated that plasma levels of LMWMs measured by 1H-NMR could be potential biomarkers associated with T2D. Moreover, alanine and valine can help in the early detection of the cardiovascular risk associated with this metabolic disease.

Lipid lowering combination therapy: From prevention to atherosclerosis plaque treatment.

Statins have contributed to the prevention of numerous atherosclerotic cardiovascular (CV) events and cardiovascular deaths in the past three decades. The benefit of statins is mainly mediated by the lowering of LDLc. According to scientific evidence, the current international guidelines recommend very low LDLc goals in patients at high/very high cardiovascular risk because they are associated with fewer CV events and improvements in atherosclerotic plaques. However, these goals often cannot be obtained with statins alone. Recent RCTs have demonstrated that these CV benefits can also be obtained with nonstatin LDLc-lowering drugs such as PCSK9 inhibitors (alirocumab and evolocumab), ezetimibe and bempedoic acid, while evidence with inclisiran is upcoming. Icosapent ethyl, a lipid metabolism modifier, has also shown an effect on event reduction. Physicians should take advantage of the currently available lipid-lowering therapies, choosing the drug or combination of drugs that is most appropriate for each patient according to his or her CV risk and baseline LDLc concentration. Strategies implementing combination therapies from early stages or even from the outset may increase the number of patients attaining LDLc goals, thereby preventing new CV episodes and improving existing atherosclerotic lesions.

Plasma Expression of Carotid Plaque Presence-Related MicroRNAs Is Associated with Inflammation in Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is associated with problems beyond the joints such as cardiovascular (CV) disease. MicroRNA-24, -146 and -Let7a are associated with carotid plaque presence in RA patients. We evaluated whether these microRNAs were involved in the inflammatory state of RA, and we studied their gene targets to understand their role in inflammation and atherosclerosis. A total of 199 patients with RA were included. Inflammatory variables such as disease activity score 28 (DAS28) and erythrocyte sedimentation rate (ESR) were quantified. MicroRNAs were extracted from plasma and quantified with qPCR. Multivariate models and classification methods were used for analysis. The multivariate models showed that diminished expression of microRNA-146 was associated with inferior levels of DAS28-ESR, and the decreased expression of microRNA-24, -146 and -Let7a were associated with lowered ESR in the overall cohort. When microRNAs were evaluated globally, a global increase was associated with increased DAS28-ESR and ESR in the overall cohort. Sex-stratified analyses showed different associations of these microRNAs with the inflammatory variables. Finally, random forest models showed that microRNAs have a pivotal role in classifying patients with high and low inflammation. Plasmatic expressions of microRNA-24, -146 and -Let7a were associated with inflammatory markers of RA. These microRNAs are associated with both inflammation and atherosclerosis and are potential therapeutic targets for RA.

PCSK9 Inhibitors Have Apolipoprotein C-III-Related Anti-Inflammatory Activity, Assessed by 1H-NMR Glycoprotein Profile in Subjects at High or very High Cardiovascular Risk.

Atherosclerosis is a chronic inflammatory disease caused by the accumulation of cholesterol in the intima. Proprotein convertase subtilisin/kexin type 9 inhibitors (iPCSK9) can reduce low-density lipoprotein (LDL) cholesterol levels by 60%, but there is still no evidence that they can lower markers of systemic inflammation such as high-sensitivity C-reactive protein (hsCRP). Acute-phase serum glycoproteins are upregulated in the liver during systemic inflammation, and their role as inflammatory biomarkers is under clinical evaluation. In this observational study, we evaluate the effects of iPCSK9 on glycoproteins (Glyc) A, B and F. Thirty-nine patients eligible for iPCSK9 therapy were enrolled. One sample before and after one to six months of iPCSK9 therapy with alirocumab was obtained from each patient. Lipids, apolipoproteins, hsCRP and PCSK9 levels were measured by biochemical analyses, and the lipoprotein and glycoprotein profiles were measured by 1H nuclear magnetic resonance (1H-NMR). The PCSK9 inhibitor reduced total (36.27%, p < 0.001), LDL (55.05%, p < 0.001) and non-high-density lipoprotein (HDL) (45.11%, p < 0.001) cholesterol, apolipoprotein (apo) C-III (10%, p < 0.001), triglycerides (9.92%, p < 0.001) and glycoprotein signals GlycA (11.97%, p < 0.001), GlycB (3.83%, p = 0.017) and GlycF (7.26%, p < 0.001). It also increased apoA-I (2.05%, p = 0.043) and HDL cholesterol levels (11.58%, p < 0.001). Circulating PCSK9 levels increased six-fold (626.28%, p < 0.001). The decrease in Glyc signals positively correlated with the decrease in triglycerides and apoC-III. In conclusion, in addition to LDL cholesterol, iPCSK9 therapy also induces a reduction in systemic inflammation measured by 1H-NMR glycoprotein signals, which correlates with a decrease in triglycerides and apoC-III.

Analysis of LDL and HDL size and number by nuclear magnetic resonance in a healthy working population: The LipoLab Study.

BACKGROUND AND AIM: Atherosclerosis is the underlying process in cardiovascular disease (CVD), the first cause of death in developed countries. We aimed to identify people with no known CVD and normal values of LDL-C and HDL-C, but with alterations in the number and size of lipoprotein particles (as measured by nuclear magnetic resonance [NMR]) and to analyse their sociodemographic, clinical and biochemical characteristics. METHODS: Cross-sectional study in occupational risks prevention centre in Castellón (Spain) in 2017 and 2018, in consecutively recruited adults (18-65 years) with no known CVD. Sociodemographic, clinical and biochemical variables were collected. Lipid profiles were analysed (Liposcale test), along with the concentration, size and number of the main types of lipoprotein particles, determined by 2D diffusion-ordered NMR spectroscopy. Using contingency tables, we analysed the characteristics of people with normal LDL and HDL cholesterol but abnormal levels of LDL and HDL particles. The magnitude of association between explanatory variables and abnormal levels of each kind of lipoprotein was assessed with multivariable logistic regression models. RESULTS: Of the 400 total participants (31.3% women; age 46.4 ± 4.3 years), 169 had normal LDL and HDL cholesterol. Abnormal lipoprotein particle values depended on the subtype: prevalence of abnormal LDL levels ranged from 8.3% to 36.7%; and of HDL, from 28.4% to 42.6%. High systolic blood pressure and total cholesterol were significantly associated with abnormal LDL levels. Male sex and high systolic blood pressure were associated with abnormalities in HDL. CONCLUSIONS: An extended lipids profile, obtained by NMR, enables the identification of people with normal HDL-C and LDL-C levels who present abnormal levels of LDL-P and/or HDL-P. Higher total cholesterol, systolic blood pressure, BMI and male sex were significantly associated with these abnormal values.

Reasons Why Combination Therapy Should Be the New Standard of Care to Achieve the LDL-Cholesterol Targets : Lipid-lowering combination therapy.

PURPOSE OF REVIEW: The aim of this report is to review the scientific evidence supporting that lipid lowering therapy (LLT), beyond statins, reduces cardiovascular risk; therefore, treatment strategies based on lipid-lowering drug combination should be implemented. RECENT FINDINGS: A strong scientific body of evidence supports the effect of statins on cardiovascular risk reduction. Recent trials using non-statin LLT, ezetimibe, and PCSK9 inhibitors have provide scientific evidence about their impact on cardiovascular prevention. Current clinical guidelines still recommend using high-intensity statin monotherapy before considering combination therapy. The causal effect of LDL-C on atherosclerosis is well established. Moreover, new RCT, meta-analysis, and Mendelian randomization data, support that the main determinant of risk reduction is the absolute LDL reduction regardless of LLT. Accordingly, the "high-intensity statin therapy" concept should be substituted by "high-intensity lipid lowering therapy." Combination therapy must become the standard of care of hypercholesterolemia treatment.

Palmitate decreases migration and proliferation and increases oxidative stress and inflammation in smooth muscle cells: role of the Nrf2 signaling pathway.

Fatty acids are essential to cell functionality and may exert diverging vascular effects including migration, proliferation, oxidative stress, and inflammation. This study examined the effect of palmitate on human coronary artery smooth muscle cell (HCASMC) function. An in vitro wound-healing assay indicated that palmitate decreased HCASMC migration in dose- and time-dependent manners. Furthermore, bromodeoxyuridine incorporation assays indicated that palmitate decreased HCASMC proliferation in a dose-response manner. Palmitate also increased reactive oxygen species formation, malondialdehyde content, and intracellular lipid droplets accompanied with increased fatty acid binding protein 4 expression. Moreover, palmitate induced gene expression (monocyte chemoattractant protein 1, matrix metalloproteinase-2, IL-1ß, IL-6, IL-8, and TNF-α) and intracellular protein content (plasminogen activator inhibitor-1 and urokinase plasminogen activator) of inflammatory mediators. Finally, we showed that palmitate activates the transcription factor Nrf2 and the upstream kinases ERK1/2 and Akt in HCASMCs. The inhibitor of Nrf2, trigonelline, significantly attenuated palmitate-induced HCASMC expression of the Nrf2 target gene NQO1. These findings indicate that palmitate might be critically related to HCASMC function by slowing cell migration and proliferation and inducing lipid-laden cells, oxidative stress, and inflammation in part by activation of the Nrf2 transcription factor. Palmitate's activation of proinflammatory Nrf2 signaling may represent a novel mechanism mediating the proatherogenic actions of saturated fatty acids.

HDL Triglycerides: A New Marker of Metabolic and Cardiovascular Risk.

While cholesterol content in high-density lipoproteins (HDLs) is a well-established inverse marker of cardiovascular risk, the importance of HDL-triglyceride (HDL-TG) concentration is not well known. We aim to examine plasma HDL-TG concentrations, assessed by 1H-NMR, in patients with metabolic diseases and their association with classical biomarkers. In this cross-sectional study, we included 502 patients with type 2 diabetes or metabolic syndrome attending the lipid unit of our University Hospital. The presence of arteriosclerotic plaques was assessed by ultrasonography. A complete lipoprotein profile was performed by 1H-NMR (Liposcale test). HDL-TG was strongly positively correlated with total triglycerides, glycerol, and fatty liver index, while a strong negative correlation was observed with HDL-cholesterol (HDL-C) and HDL-particle number (HDL-P). HDL-TG was associated with all triglyceride-rich lipoprotein parameters and had an opposite association with HDL-C and HDL-P. It was also significantly correlated with circulating cholesterol ester transfer protein (CETP). HDL-TG concentrations were higher as metabolic syndrome components increased. HDL-TG was also higher with worsening glucose metabolism. Patients with carotid plaques also showed higher HDL-TG. In contrast to HDL-C, HDL-TG is directly associated with metabolism and arteriosclerotic vascular alterations. HDL-TG should be considered a biomarker of metabolic and cardiovascular risk and could be a marker of HDL dysfunction.

Characterization of 1H NMR Plasma Glycoproteins as a New Strategy To Identify Inflammatory Patterns in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease associated with a high index of morbidity and mortality from cardiovascular diseases. We used 1H NMR to characterize the plasma glycoprotein and lipoprotein profiles of a cohort of patients with RA ( n = 210) versus healthy individuals ( n = 203) to associate them with the RA disease and its severity. Using 1H NMR, we developed a line-shape method to characterize the two peaks associated with glycoproteins (GlycA and GlycB) and its derived variables: areas of GlycB (Area GlycB) and GlycA (Area GlycA), shape factors of these two peaks (H/W = height/width), and the distance between them (Distance GlycB-GlycA). We also used the advanced lipoprotein test Liposcale (CE) to characterize the lipoprotein subclasses. The standard lipid panel and traditional inflammatory markers such as C-reactive protein, the erythrocyte sedimentation rate, fibrinogen, the rheumatoid factor, anticitrullinated peptide antibodies, and the DAS28 index have also been determined. RA patients presented a significant 10.65% increase in the GlycA associated area compared with the control group ( p = 2.21 × 10-10). They also presented significantly higher H/W GlycA and GlycB ratios than the control population (H/W GlycB p = 7.88 × 10-8; H/W GlycA p = 5.61 × 10-8). The prediction model that uses the traditional inflammatory variables and the 1H NMR-derived parameters presented an AUC that was almost 10% higher than the model that only uses the traditional inflammatory variables (from 0.7 to 0.79 AUC). We have demonstrated that GlycA and GlycB variables derived from 1H NMR, along with classic inflammatory parameters, help to improve the classification of individuals with high RA disease activity.

FABP4 inhibitor BMS309403 decreases saturated-fatty-acid-induced endoplasmic reticulum stress-associated inflammation in skeletal muscle by reducing p38 MAPK activation.

AIMS: Fatty acid binding protein 4 (FABP4) inhibitors have been proposed as potential therapeutic approaches against insulin resistance-related inflammation and type 2 diabetes mellitus. However, the underlying molecular mechanisms by which these molecules drive these effects in skeletal muscle remain unknown. Here, we assessed whether the FABP4 inhibitor BMS309403 prevented lipid-induced endoplasmic reticulum (ER) stress-associated inflammation in skeletal muscle. MATERIALS AND METHODS: The BMS309403 treatment was assessed both in the skeletal muscle of high-fat diet (HFD)-fed mice and in palmitate-stimulated C2C12 myotubes. RESULTS: HFD feeding promoted insulin resistance, which is characterized by increased plasma levels of glucose, insulin, non-esterified fatty acids, triglycerides, resistin, and leptin and reduced plasma levels of adiponectin compared with control mice fed a standard diet. Additionally, insulin-resistant animals showed increased FABP4 plasma levels. In line with this evidence, recombinant FABP4 attenuated the insulin-induced AKT phosphorylation in C2C12 myotubes. Treatment with BMS309403 reduced lipid-induced ER stress and inflammation in both mouse skeletal muscle and C2C12 myotubes. The effects of the FABP4 inhibitor reducing lipid-induced ER stress-associated inflammation were related to the reduction of fatty acid-induced intramyocellular lipid deposits, ROS and nuclear factor-kappaB (NF-κB) nuclear translocation. Accordingly, BMS309403 reduced lipid-induced p38 MAPK phosphorylation, which is upstream of NF-κB activation. CONCLUSION: Overall, these findings indicate that BMS309403 reduces fatty acid-induced ER stress-associated inflammation in skeletal muscle by reducing p38 MAPK activation.

Exogenous FABP4 increases breast cancer cell proliferation and activates the expression of fatty acid transport proteins.

Adipose tissue plays an important role in tumor progression, because it provides nutrients and adipokines to proliferating cells. Fatty acid binding protein 4 (FABP4) is a key adipokine for fatty acid transport. In metabolic pathologies, plasma levels of FABP4 are increased. However, the role of this circulating protein is unknown. Recent studies have demonstrated that FABP4 might have a role in tumor progression, but the molecular mechanisms involved are still unclear. In this study, we analysed the role of eFABP4 (exogenous FABP4) in breast cancer progression. MCF-7 and MDA-MB-231 breast cancer cells did not express substantial levels of FABP4 protein, but intracellular FABP4 levels increased after eFABP4 incubation. Moreover, eFABP4 enhanced the proliferation of these breast cancer cells but did not have any effect on MCF-7 and MDA-MB-231 cell migration. Additionally, eFABP4 induced the AKT and MAPK signaling cascades in breast cancer cells, and the inhibition of these pathways reduced the eFBAP4-mediated cell proliferation. Interestingly, eFABP4 treatment in MCF-7 cells increased levels of the transcription factor FoxM1 and the fatty acid transport proteins CD36 and FABP5. In summary, we showed that eFABP4 plays a key role in tumor proliferation and activates the expression of fatty acid transport proteins in MCF-7 breast cancer cells. © 2016 Wiley Periodicals, Inc.

New insights into circulating FABP4: Interaction with cytokeratin 1 on endothelial cell membranes.

Fatty acid-binding protein 4 (FABP4) is an adipose tissue-secreted adipokine that is involved in the regulation of energetic metabolism and inflammation. Increased levels of circulating FABP4 have been detected in individuals with cardiovascular risk factors. Recent studies have demonstrated that FABP4 has a direct effect on peripheral tissues, specifically promoting vascular dysfunction; however, its mechanism of action is unknown. The objective of this work was to assess the specific interactions between exogenous FABP4 and the plasma membranes of endothelial cells. Immunofluorescence assays showed that exogenous FABP4 localized along the plasma membranes of human umbilical vein endothelial cells (HUVECs), interacting specifically with plasma membrane proteins. Anti-FABP4 immunoblotting revealed two covalent protein complexes containing FABP4 and its putative receptor; these complexes were approximately 108 kDa and 77 kDa in size. Proteomics and mass spectrometry experiments revealed that cytokeratin 1 (CK1) was the FABP4-binding protein. An anti-CK1 immunoblot confirmed the presence of CK1. FABP4-CK1 complexes were also detected in HAECs, HCASMCs, HepG2 cells and THP-1 cells. Pharmacological FABP4 inhibition by BMS309403 results in a slight decrease in the formation of these complexes, indicating that fatty acids may play a role in FABP4 functionality. In addition, we demonstrated that exogenous FABP4 crosses the plasma membrane to enter the cytoplasm and nucleus in HUVECs. These findings indicate that exogenous FABP4 interacts with plasma membrane proteins, specifically CK1. These data contribute to our current knowledge regarding the mechanism of action of circulating FABP4.

APOA5 genetic and epigenetic variability jointly regulate circulating triacylglycerol levels.

Apolipoprotein A5 gene (APOA5) variability explains part of the individual's predisposition to hypertriacylglycerolaemia (HTG). Such predisposition has an inherited component (polymorphisms) and an acquired component regulated by the environment (epigenetic modifications). We hypothesize that the integrated analysis of both components will improve our capacity to estimate APOA5 contribution to HTG. We followed a recruit-by-genotype strategy to study a population composed of 44 individuals with high cardiovascular disease risk selected as being carriers of at least one APOA5 SNP (-1131T>C and/or, S19W and/or 724C>G) compared against 34 individuals wild-type (WT) for these SNPs. DNA methylation patterns of three APOA5 regions [promoter, exon 2 and CpG island (CGI) in exon 3] were evaluated using pyrosequencing technology. Carriers of APOA5 SNPs had an average of 57.5% higher circulating triacylglycerol (TG) levels (P=0.039). APOA5 promoter and exon 3 were hypermethylated whereas exon 2 was hypomethylated. Exon 3 methylation positively correlated with TG concentration (r=0.359, P=0.003) and with a lipoprotein profile associated with atherogenic dyslipidaemia. The highest TG concentrations were found in carriers of at least one SNP and with a methylation percentage in exon 3 ≥82% (P=0.009). In conclusion, CGI methylation in exon 3 of APOA5 acts, in combination with -1131T>C, S19W and 724C>G polymorphisms, in the individual's predisposition to high circulating TG levels. This serves as an example that combined analysis of SNPs and methylation applied to a larger set of genes would improve our understanding of predisposition to HTG.

Liposcale: a novel advanced lipoprotein test based on 2D diffusion-ordered 1H NMR spectroscopy.

Determination of lipoprotein particle size and number using advanced lipoprotein tests (ALTs) is of particular importance to improve cardiovascular risk prediction. Here we present the Liposcale test, a novel ALT based on 2D diffusion-ordered (1)H NMR spectroscopy. Our method uses diffusion coefficients to provide a direct measure of the mean particle sizes and numbers. Using 177 plasma samples from healthy individuals and the concentration of ApoB and ApoA from isolated lipoprotein fractions, our test showed a stronger correlation between the NMR-derived lipoprotein particle numbers and apolipoprotein concentrations than the LipoProfile(®) test commercialized by Liposcience. We also converted LDL particle numbers to ApoB equivalents (milligrams per deciliter) and our test yielded similar values of LDL-ApoB to the LipoProfile(®) test (absolute mean bias of 8.5 and 7.4 mg/dl, respectively). In addition, our HDL particle number values were more concordant with the calibrated values determined recently using ion mobility. Finally, principal component analysis distinguished type 2 diabetic patients with and without atherogenic dyslipidemia (AD) on a second cohort of 307 subjects characterized using the Liposcale test (area under the curve = 0.88) and showed concordant relationships between variables explaining AD. Altogether, our method provides reproducible and reliable characterization of lipoprotein particles and it is applicable to pathological states such as AD.

Circulating PCSK9 levels are positively correlated with NMR-assessed atherogenic dyslipidaemia in patients with high cardiovascular risk.

The proprotein convertase subtilisin/kexin type 9 (PCSK9) gene regulates cholesterol homoeostasis by accelerating low-density lipoprotein receptor (LDLR) degradation resulting in the decreased catabolism of low-density lipoprotein (LDL) leading to hypercholesterolaemia. PCSK9 has also been related to other metabolic risk factors such as triglycerides (TGs) and glucose levels and body mass index (BMI). Therefore, our aim was to study the relationship between the PCSK9 and the lipid and lipoprotein profile. We studied 267 diabetic and metabolic syndrome patients who were not receiving any lipid-lowering therapy. We measured circulating lipids, cholesterol in remnant lipoproteins (RLPc) and PCSK9 levels. A detailed lipoprotein profile was determined based on NMR. Plasma PCSK9 levels were significantly and positively correlated with TG (r=0.136, P=0.033), total cholesterol (r=0.219, P<0.001) and apoB (apolipoprotein B; r=0.226, P=0.006) circulating levels and with an atherogenic profile of lipoprotein subclasses. In further detail, circulating PCSK9 levels were positively correlated with large very-low density lipoprotein (VLDL) particles, (r=0.210, P=0.001) and with their remnants, the intermediate-density lipoprotein (IDL) particles (r=0.206, P=0.001); positively correlated with smaller LDL particles (for small LDL: r=0.224, P<0.001; for medium small LDL: r=0.235, P<0.001; and for very small LDL: r=0.220, P<0.001); and with high-density lipoprotein (HDL) particles (r=0.146, P<0.001), which is mainly explained by the PCSK9 correlation with the smallest HDL particles (r=0.130, P=0.037). In addition, circulating PCSK9 levels were positively correlated with the pro-atherogenic circulating RLPc levels (r=0.171, P=0.006). All of the correlations were adjusted by age, gender and BMI. PCSK9 levels are significantly and positively correlated with atherogenic lipoproteins such as large VLDL, IDL, the smallest LDL, the smallest HDL particles and RLPc levels.