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ATPase inhibitory factor 1 (IF1) is a 9.5 kDa protein that binds to mitochondrial and plasma membrane ATP synthase and selectively inhibits ATP hydrolysis. Recently, IF1 was identified in systemic circulation in humans. IF1 appeared as an independent determinant of HDL-cholesterol with lower levels in coronary heart disease (CHD) patients. Moreover, IF1 was also found to negatively associate with mortality in these patients, supporting the notion that circulating IF1 could be a promising biomarker of cardiovascular disease. However, in previous studies, IF1 was quantified by a non-standardized competitive enzyme-linked immunosorbent assay (ELISA). Herein, we have validated a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) enabling the accurate quantification of IF1 in human plasma. Plasma IF1 was trypsin-digested through an optimized procedure before LC-MS/MS analysis. The method was successfully validated over 4 independent experiments into the range of 100-1500 ng/mL. Intra- and inter-assay variation coefficients had never exceeded 14.2% and accuracy ranged between 95% and 102% for the selected EAGGAFGK peptide marker. Subsequently, the results of the LC-MS/MS method were compared with those obtained using ELISA in 204 individuals from the GENES study. We found that IF1 plasma levels obtained using both techniques were strongly correlated (r = 0.89, p < 0.0001), while the Bland-Altman plot did not indicate any major statistically significant differences. To clinically validate LC-MS/MS, we confirmed the positive correlation between IF1 plasma levels and HDL-cholesterol (r = 0.38, p < 0.0001). Besides, we found lower IF1 plasma levels in CHD patients compared to controls (431 ± 132 ng/mL and 555 ± 173 ng/mL, respectively; p < 0.0001). Hence, it can be concluded that the presented LC-MS/MS analytical method provides a highly specific strategy for IF1 quantification in human plasma and could be proposed as a reference method., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The atheroprotective property of High Density Lipoprotein (HDL) is supported by many epidemiological studies and cellular and in vivo approaches on animal models. While the anti-atherogenic effects of HDL are thought to derive primarily from its role in reverse cholesterol transport, together with anti-inflammatory, anti-oxidant, anti-thrombotic and cytoprotective properties, the mechanisms that support these effects are still not completely understood. However, many advances in identifying the cellular partners involved in HDL functions have been made over the last two decades. This review highlights the diverse roles of the HDL receptor ecto-F1-ATPase coupled to purinergic P2Y receptors in the modulation of important metabolic and vascular functions of HDL. On hepatocytes, the ecto-F1-ATPase is coupled to P2Y13 receptor and contributes to HDL holoparticle endocytosis. On endothelial cells, ecto-F1-ATPase/P2Ys pathway is involved in HDL-mediated endothelial protection and HDL transcytosis. The clinical relevance of this F1-ATPase/P2Ys axis in humans has recently been supported by the identification of serum F1-ATPase inhibitor (IF1) as an independent determinant of HDL-Cholesterol (HDL-C) and coronary heart disease risk. Therapeutic strategies targeting F1-ATPase/P2Y pathways for the treatment of atherosclerosis are currently being explored., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

HDL is strongly inversely related to cardiovascular risk. Hepatic HDL uptake is controlled by ecto-F1-ATPase activity, and potentially inhibited by mitochondrial inhibitor factor 1 (IF1). We recently found that IF1 is present in serum and correlates with HDL-cholesterol (HDL-C). Here, we have evaluated the relationship between circulating IF1 and plasma lipoproteins, and we determined whether IF1 concentration is associated with the risk of coronary heart disease (CHD). Serum IF1 was measured in 648 coronary patients ages 45-74 and in 669 matched male controls, in the context of a cross-sectional study on CHD. Cardiovascular risk factors were documented for each participant, including life-style habits and biological and clinical markers. In controls, multivariate analysis demonstrated that IF1 was independently positively associated with HDL-C and apoA-I (r = 0.27 and 0.28, respectively, P < 0.001) and negatively with triglycerides (r = -0.23, P < 0.001). Mean IF1 concentration was lower in CHD patients than in controls (0.43 mg/l and 0.53 mg/l, respectively, P < 0.001). In multivariate analyses, following adjustments on cardiovascular risk factors or markers, IF1 was negatively related to CHD (P < 0.001). This relationship was maintained after adjustment for HDL-C or apoA-I. This study identifies IF1 as a new determinant of HDL-C that is inversely associated with CHD.

Exosomes are bioactive vesicles released from multivesicular bodies (MVB) by intact cells and participate in intercellular signaling. We investigated the presence of lipid-related proteins and bioactive lipids in RBL-2H3 exosomes. Besides a phospholipid scramblase and a fatty acid binding protein, the exosomes contained the whole set of phospholipases (A2, C, and D) together with interacting proteins such as aldolase A and Hsp 70. They also contained the phospholipase D (PLD) / phosphatidate phosphatase 1 (PAP1) pathway leading to the formation of diglycerides. RBL-2H3 exosomes also carried members of the three phospholipase A2 classes: the calcium-dependent cPLA(2)-IVA, the calcium-independent iPLA(2)-VIA, and the secreted sPLA(2)-IIA and V. Remarkably, almost all members of the Ras GTPase superfamily were present, and incubation of exosomes with GTPgammaS triggered activation of phospholipase A(2) (PLA(2))and PLD(2). A large panel of free fatty acids, including arachidonic acid (AA) and derivatives such as prostaglandin E(2) (PGE(2)) and 15-deoxy-Delta(12,14)-prostaglandinJ(2) (15-d PGJ(2)), were detected. We observed that the exosomes were internalized by resting and activated RBL cells and that they accumulated in an endosomal compartment. Endosomal concentrations were in the micromolar range for prostaglandins; i.e., concentrations able to trigger prostaglandin-dependent biological responses. Therefore exosomes are carriers of GTP-activatable phospholipases and lipid mediators from cell to cell.

The preovulatory human follicular fluid contains only HDLs as a lipoprotein class with a typically high proportion of prebeta HDL. We first examined the role of follicular fluid and HDL subfractions on human spermatozoa capacitation, a process characterized by a hyperactivation of the flagellar movement and a depletion of plasma membrane cholesterol. Whole follicular fluid and isolated HDL, used at constant free cholesterol concentration, were both able to promote an early flagellar hyperactivation. Moreover, incubation of [(3)H]cholesterol-labeled spermatozoa with follicular fluid induced a rapid cholesterol efflux from spermatozoa that was confirmed by mass measurements of cholesterol transfer. Using isolated HDL, the cholesterol efflux had a similar time course and represented 70% of that mediated by whole follicular fluid. We then analyzed the time course of radioactive labeling of HDL subfractions. In the first minute of incubation, we found that the prebeta HDL fraction incorporated the main part of the radioactivity (60%), with the rest being found in alpha-HDL, but strikingly, the labeling of alpha-HDL increased with time at the expense of prebeta HDL.Thus, our results indicate that HDLs are involved in both spermatozoa hyperactivation and cholesterol effl ux and suggest the role of prebeta-HDL particles as fi rst cellular cholesterol acceptors.

Although the iron-heart disease hypothesis is prevalent, the epidemiological findings are incongruent. The relationship of serum ferritin with early cardiovascular disease (CVD), particularly atherosclerosis, has not been evaluated extensively, particularly with accounting for inflammation. We examined this association in a case-control study of 124 age- and sex-matched pairs embedded in the population-based random sample (MONICA survey) in Southwest France, taking into account inflammation status. Cases had >or=2 carotid atherosclerotic plaques and controls had none. Inflammation was assessed using several markers, including serum alpha-1 acid glycoprotein (AGP) and high sensitivity C-reactive protein. There was an interaction of inflammation with group (case/control) for serum ferritin. In adults without elevated AGP, serum ferritin was significantly greater in atherosclerotic cases than in adults in the control group. In models adjusted for CVD risk factors, the odds of atherosclerosis increased with the increase in serum ferritin in individuals without elevated AGP; for every 10-microg/L increase in serum ferritin, the risk for atherosclerosis increased by 3% (odds ratio [95% CI]: 1.03 [1.01-1.06]). In conclusion, carotid atherosclerosis was positively associated with serum ferritin in individuals free from subclinical inflammation based on AGP. Further prospective and/or experimental studies are needed to corroborate the observed association of iron status with atherosclerosis.

Background: Reports about the relationships between insulin concentrations and CHD risk are controversial. The objective of this survey was to study the association between insulin levels and CHD risk in middle-aged male participants of the PRIME Study after 5 years of follow-up., Methods: Our study adopted a nested case-control design including 294 cases of CHD and 536 controls randomly selected among healthy participants from the PRIME cohort. Data were obtained by questionnaires (medical history, lifestyle), standardised clinical measurements (blood pressure, anthropometric measurements), and a blood sample was obtained for biological measurements. Odds-Ratios for associations of four ordered classes of insulin concentration with CHD risk after adjustment for confounding factors were estimated using conditional logistic regression., Results: In Belfast, a significant trend (p<0.03) was observed between insulin classes and CHD risk in bivariate analyses, but this association lost its significance after multiple adjustments. In the French centres, a high risk of CHD (OR=3.24 [1.80-5.85], p<0.0001) was observed only for the second class of insulin concentration (6.5 to 9.9 mIU/l), compared with the reference class (<6.5 mIU/l). After multiple adjustments, this association remained highly significant (OR=2.92 [1.44-5.92], p<0.005)., Conclusions: In Belfast (high-risk population), a significant trend was observed between insulin concentration classes and CHD risk but hyperinsulinaemia lost its association with CHD risk in multivariate analyses. In the French centres (lower risk population), slightly increased insulin concentrations were associated with a high risk of CHD, independently of cardiovascular risk factors and other features of the metabolic syndrome, but very high insulin concentrations were not.

Lysophosphatidic acid (LPA) is present in abundance in serum resulting from platelet activation and is also found in other biological fluids. LPA controls numerous cellular responses and plays a role in specific functions such as wound healing, especially in the skin. Nevertheless, its presence in the skin has never been investigated. Since re-epithelialization occurs after blister rupture, we tested the presence of endogenous LPA in blister fluid and investigated a possible mechanism for its biosynthesis and biological functions. Using a radioenzymatic assay, LPA was detected in 33 blister fluids originating from 24 bullous dermatoses, and at higher concentrations than in plasma. In parallel, blister fluids contained a lysophospholipase D (LPLD) activity but no detectable phospholipase A2 activity. The expressions of the LPLD autotaxin (ATX) and of LPA1-receptor (LPA1-R) were greatly increased in blister skin when compared with normal skin. Finally, LPA was found to have a positive effect on the migration of cultured keratinocytes. These results show that LPA is present in blister fluid synthesized by the LPLD ATX. Due to its ability to enhance keratinocyte migration, LPA in blister fluid could, via the LPA1-R, play an important role in re-epithelialization occurring after blister rupture.

This study aimed to assess endothelium-dependent vasomotor function in non-culprit coronary vessels in patients with recent acute coronary syndrome (ACS). Endothelial dysfunction can also concern peripheral vessels during ACS, but the frequency of this phenomenon at coronary circulation level is unknown. Endothelial function was assessed by infusion of graded doses of acetylcholine (ACh) in a non-culprit coronary artery of normal appearance in 43 patients having recently suffered from non-ST ACS, and reassessed 6 months later. Endothelium-dependent vasoreactivity was impaired at baseline in 81% (35/43) of the patients, and only 19% (8/43) of patients showed a normal response to ACh. Among the 35 subjects with initial dysfunction, 77% showed a significant improvement 6 months later. All patients without initial endothelial dysfunction remained normal. C-reactive protein (CRP) level was elevated at month 0, and had declined at follow-up, tending towards normal levels. At that time, apolipoprotein A-I (apoA-I) levels were correlated with vasomotor improvement in univariate (p < 0.02) and multivariate analysis (p < 0.04). In conclusion, endothelium dysfunction is very frequently observed after recent ACS in non-culprit and angiographically normal coronary arteries, and an improvement occurs within 6 months in most cases. After resolution of the initial inflammation, apoA-I seems to play an important role in endothelial function.

The prevalence of coronary heart disease is much higher in men than in women and sex hormones might play a role in these differences through their influence on the lipid profile. The aim of this cross-sectional study was to study the relationship between hormonal markers (total testosterone (TT), estradiol (E2), sex-hormone-binding globulin (SHBG)) and plasma lipids in a population-based sample. Subjects were 352 men, 50-59 years old, selected in France (Lille, Strasbourg and Toulouse) and Northern-Ireland (Belfast) who had questionnaires and a medical examination at baseline of the PRIME prospective study (1991-1993). Pearson correlation coefficients and Student's t tests were used to identify factors associated with plasma lipids. Multiple linear regression models were used for multivariate analyses, using triglycerides (TG) (log-transformed) and high density-lipoprotein cholesterol (HDL-C) as dependent variables. SHBG and TT were negatively correlated with TG (p<0.0001 and p<0.05, respectively) and positively correlated with HDL-C (p<0.0001 and p<0.01). E2 was positively correlated with TG (p<0.05). No significant association was found between sex-hormones and LDL-C. In multiple linear regression analyses, SHBG remained independently associated negatively with TG (p<0.01) and positively with HDL-C (p<0.0001) after adjustment for centre of recruitment, age, body mass index, systolic blood pressure, smoking, alcohol intake and physical activity. After further adjustment for insulin, the association between SHBG and HDL-C remained highly significant (p<0.0001). The association between SHBG and TG was weakened but remained also significant. Our results suggest that SHBG might to be a central protein in the hormonal regulation of the lipid profile.

The involvement of phosphoinositide 3-kinase C2alpha in vascular smooth muscle cell migration was investigated. Products of phosphoinositide 3-kinase, phosphatidylinositol-3-phosphate, and phosphatidylinositol-3,4-bis-phosphate were increased upon smooth muscle cell migration but their synthesis was affected only partially by phosphoinositide 3-kinase inhibitors, wortmannin and LY-294002. Using specific antibody, we showed that the wortmannin/LY-294002 poorly sensitive phosphoinositide 3-kinase C2alpha is expressed in smooth muscle cells. Measurement of phosphoinositide 3-kinase C2alpha activity in vitro, after immunoprecipitation, clearly demonstrated its activation upon smooth muscle cell migration. Moreover, for the first time, phosphoinositide 3-kinase C2alpha was found to be differentially regulated by alpha(v)beta(3) and alpha(v)beta(5) integrin engagement. Finally, we have identified two new potential phosphoinositide 3-kinase C2alpha-binding proteins, p70 and p110, which both may be tyrosine phosphorylated. Thus, phosphoinositide 3-kinase C2alpha might represent a new regulatory pathway of cell migration downstream of integrin engagement.

Hepatic lipase (HL) is a lipolytic enzyme, synthesized by hepatocytes and found localized at the surface of liver sinusoid capillaries. In humans, the enzyme is mostly bound onto heparan-sulfate proteoglycans at the surface of hepatocytes and also of sinusoid endothelial cells. HL shares a number of functional domains with lipoprotein lipase and with other members of the lipase gene family. It is a secreted glycoprotein, and remodelling of the N-linked oligosaccharides appears to be crucial for the secretion process, rather than for the acquisition of the catalytic activity. HL is also present in adrenals and ovaries, where it might promote delivery of lipoprotein cholesterol for steroidogenesis. However, evidence of a local synthesis is still controversial. HL activity is fairly regulated according to the cell cholesterol content and to the hormonal status. Coordinate regulations have been reported for both HL and the scavenger-receptor B-I, suggesting complementary roles in cholesterol metabolism. However, genetic variants largely contribute to HL variability and their possible impact in the development of a dyslipidemic phenotype, or in a context of insulin-resistance, is discussed.

Phospholipase B (PLB) is an enzyme that displays both phospholipase A(2) and lysophospholipase activities. Analysis of human epidermis homogenates indicated the presence of a 97 kDa PLB protein, as well as a phospholipase A(2) activity, both being enriched in the soluble fraction. Immunolabelling and in situ hybridization experiments showed that this enzyme is expressed in the different layers of epidermis with an accumulation at the dermo-epidermis junction. RT-PCR data indicated that PLB is specifically expressed in natural and reconstructed epidermis. By 3'-RACE-PCR and screening of human genome databases, we obtained a 3600 bp cDNA coding for human PLB highly homologous to already described intestinal brush border PLBs. These data led us to conclude that the soluble PLB corresponds to a proteolytic cleavage of the membrane anchored protein. Altogether, our results provide the first characterization of human PLB which should play an important role in epidermal barrier function.

This study reports a single-step analysis of the molecular species of endogenous ceramides and of the ceramide moiety of sphingomyelins in biological samples, using gas liquid chromatography (GLC). Silylated sphingomyelins were quantitatively converted to monosilylated ceramide upon injection into GLC, whereas the free ceramides were di-silylated on the primary and secondary alcohol function, as confirmed by mass spectrometry. The reproducible shift of the retention times between the mono- and di-silylated derivatives enables simultaneous quantification of the variety of sphingomyelin and ceramide molecular species. Overlapping diacylglycerols were first removed by a mild alkaline treatment of the lipid extract. The lowest detection limit (5 pmol) did not allow for identification of free ceramides in human plasma, but 17 molecular species of ceramides derived from sphingomyelins were quantified, from NC16:0 up to NC24:1. By contrast, three major free ceramides (NC16:0, NC24:0, and NC24:1) were quantified in HEPG2 and Chinese hamster ovary (CHO) cells. Upon induction of apoptosis in CHO cells by C6-ceramide, we could follow the disappearance of the C6-ceramide, its partial conversion to C6-sphingomyelin, and the prominent increase of NC16:0 ceramide. Thus, our method represents a unique procedure of simultaneous analysis of sphingomyelin and ceramide molecular species able to monitor the variation of the different pools in biological samples.