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Biomedical subjects

B Perret

Publications and source records attributed to B Perret.

At least 37 records · Page 2Linked to original sources
PubMed1999

Angiotensin I-converting enzyme gene polymorphism in a low-risk European population for coronary artery disease.

An insertion/deletion (I/D) polymorphism of the angiotensin I-converting enzyme (ACE) has been associated with an increased risk of coronary artery disease (CAD) and myocardial infarction (MI). However, this finding has not been fully investigated in European populations with very low CAD risk. In a case-control study on a population from Southern Europe (Toulouse, France), we evaluated the ACE I/D polymorphism in 405 men, aged 35-65 years, who underwent coronary angiography and in 357 representative control men within the same age range. We also explored associations in the patients between this polymorphism and CAD severity. The ACE genotype was not associated with the presence of either CAD or MI. The ACE genotype was not a marker for angiographically assessed CAD severity. In a sample in one of the European populations with the lowest CAD risk, ACE I/D polymorphism was not associated with an increased risk for CAD or MI and did not influence the extent of CAD.

Adult↗
PubMed1998

Remnant high density lipoprotein2 particles produced by hepatic lipase display high-affinity binding and increased endocytosis into a human hepatoma cell line (HEPG2).

We had previously shown that hepatic lipase plays a prominent role in promoting the generation of pre-beta HDL particles from triglyceride rich HDL2, leaving an alpha-HDL particle of decreased size that was named "remnant HDL2" [Barrans, A., et al. (1994) J. Biol. Chem. 269, 11572-11577]. Interestingly, this remnant HDL2 was rapidly cleared by the liver, suggesting a particularly high affinity of those remnant HDL2 for liver cells. In the present study, we attempted to characterize the interaction of remnant HDL2 with HepG2 cells, as compared to those of native triglyceride rich HDL2. Two main observations were made. First, while triglyceride rich HDL2 particles were able to bind only the low-affinity binding sites, the remaining particle generated after hepatic lipase lipolysis the remnant HDL2 was further able to bind to the high-affinity binding sites. Competition experiments indicate that these two remnant HDL2 binding sites were the same as the two HDL3 binding sites previously described [Barbaras, R., et al. (1994) Biochemistry 33, 2335-2340]. This is the first observation on the remodeling dependence of HDL binding onto hepatocytes. Second, following binding on those two binding sites, the remnant HDL2 were faster internalized and in higher amounts than the native triglyceride rich HDL2. All together, these observations suggest that the continuous remodeling of HDL induces different binding and internalization characteristics of the HDL particles and that the high-affinity HDL binding sites might trigger the internalization of apo HDL through the low-affinity binding sites.

Binding, Competitive↗
PubMed1998

Lipid products of phosphoinositide 3-kinase and phosphatidylinositol 4',5'-bisphosphate are both required for ADP-dependent platelet spreading.

We have shown previously that ADP released upon platelet adhesion mediated by alphaIIb beta3 integrin triggers accumulation of phosphatidylinositol 3',4'-bisphosphate (PtdIns-3,4-P2) (Gironcel, D. , Racaud-Sultan, C., Payrastre, B., Haricot, M., Borchert, G., Kieffer, N., Breton, M., and Chap, H. (1996) FEBS Lett. 389, 253-256). ADP has also been involved in platelet spreading. Therefore, in order to study a possible role of phosphoinositide 3-kinase in platelet morphological changes following adhesion, human platelets were pretreated with specific phosphoinositide 3-kinase inhibitors LY294002 and wortmannin. Under conditions where PtdIns-3, 4-P2 synthesis was totally inhibited (25 microM LY294002 or 100 nM wortmannin), platelets adhered to the fibrinogen matrix, extended pseudopodia, but did not spread. Moreover, addition of ADP to the medium did not reverse the inhibitory effects of phosphoinositide 3-kinase inhibitors on platelet spreading. Although synthetic dipalmitoyl PtdIns-3,4-P2 and dipalmitoyl phosphatidylinositol 3',4', 5'-trisphosphate restored only partially platelet spreading, phosphatidylinositol 4',5'-bisphosphate (PtdIns-4,5-P2) was able to trigger full spreading of wortmannin-treated adherent platelets. Following 32P labeling of intact platelets, the recovery of [32P]PtdIns-4,5-P2 in anti-talin immunoprecipitates from adherent platelets was found to be decreased upon treatment by wortmannin. These results suggest that the lipid products of phosphoinositide 3-kinase are required but not sufficient for ADP-induced spreading of adherent platelets and that PtdIns-4,5-P2 could be a downstream messenger of this signaling pathway.

Adenosine Diphosphate↗
PubMed1998

Phosphatidylinositol 3-kinase inhibitors block aortic smooth muscle cell proliferation in mid-late G1 phase: effect on cyclin-dependent kinase 2 and the inhibitory protein p27KIP1.

In the present study, we investigated the involvement of phosphatidylinositol 3-kinase (PI 3-kinase) activity in the progression of vascular smooth muscle cells (VSMCs) throughout the G1 phase of cell cycle. Addition of two selective inhibitors of PI 3-kinase, LY 294002 or wortmannin, to quiescent VSMCs prevented serum-induced DNA synthesis in a dose-dependent manner with IC50 of 8.7 +/- 2.0 microM and 53.9 +/- 8.5 nM, respectively. Time course studies revealed that the two PI 3-kinase inhibitors blocked VSMC proliferation in mid-late G1 phase, about 6 h before the G1/S transition. This G1 growth arrest was due, at least in part, to the reduction of the CDK2 associated kinase activity resulting mainly from the upregulation of the inhibitory protein p27KIP1.

Androstadienes↗
PubMed1998

[Prevalence and management of major risk factors in 500 men with stable coronary disease].

Management of major risk factors (smoking, hypercholesterolaemia, hypertension), in the context of secondary prevention, has an impact on recurrence and life expectancy. However, there is a delay between the publication of therapeutic trials and their diffusion within the medical community. The objective of this study was to evaluate the prevalence and management of the main risk factors in a sample of 500 men, with a mean age of 55.1 +/- 7.5 years, presenting with stable coronary artery disease. 11% of subjects had a family history of premature myocardial infarction. Smoking was frequent: 21% of smokers, 60% of ex-smokers. Hypercholesterolaemia (LDL-C > 3.4 mmole/l or treatment) was present in 82% of subjects. Only 45% of treated subjects had an LDL-C < 3.4 mmole/l. Hypertension (systolic blood pressure > or = 140 mmHg or diastolic blood pressure > or = 90 mmHg or treatment) was present in 61% of subjects. Only 33% of treated subjects were controlled. Non-insulin-dependent diabetes mellitus (blood glucose > or = 7.7 mmole/l or treatment) was present in 21% of subjects. Only 43% of treated subjects were controlled. Calculation of the distribution of major risk factors (smoking, pathological obesity, hypercholesterolaemia, hypertension, diabetes) showed that 90% of coronary patients presented at least two risk factors. Overall, the prevalence and management of risk factors in patients with stable coronary artery disease is far from optimal.

Adult↗
PubMed1997

Impaired secretion of heart lipoprotein lipase in cyclophosphamide-treated rabbit.

Cyclophosphamide administration into fasted rabbits induces a hypertriglyceridaemia and a defect in vascular lipoprotein lipase. Heart LPL activity was more than 50% decreased after antimitotic treatment in fasted animals. The tissue distribution of lipoprotein lipase activity was followed in heart using recycling perfusion. Cyclophosphamide administration resulted in a profound decline in the heparin-releasable lipoprotein lipase activity, concordant with a higher recovery in the residual heart tissue. The effects were more pronounced in fasted than in fed animals. In agreement, the proportion of neosynthesized [35S]methionine-labelled lipoprotein lipase released by heparin was decreased by 50% following antimitotic treatment. The lipolysis of very low density lipoprotein-labelled triacylglycerols was found 2.5-fold reduced in hearts from cyclophosphamide-treated rabbits as compared to controls. These results suggest that a defective secretion of lipoprotein lipase may contribute to the poor expression of lipolytic activity in the vascular bed and to the occurrence of hypertriglyceridaemia during cyclophosphamide treatment.

Animals↗
PubMed1997

Structural and functional comparison of HDL from homologous human plasma and follicular fluid. A model for extravascular fluid.

In the preovulatory period, follicular fluid contains only HDL. Biochemical characterization of such lipoproteins showed that follicular fluid HDLs were cholesterol-poor particles compared with serum HDLs, whereas the amount of phospholipids, expressed as percent weight, was significantly higher in follicular fluid HDLs (28.5%) than in serum HDLs (25.0%, P < .05). The amount of apolipoprotein (apo) A-IV per apo A-I was significantly higher in follicular fluid than in serum (0.77 versus 0.58 mg/g apo A-I, P < .02). To explore the role of HDLs as cholesterol acceptors in physiological media, we compared the ability of either whole human follicular fluids or homologous sera to promote cellular cholesterol efflux using Fu5AH rat hepatoma cells. At equivalent concentrations of HDL cholesterol in follicular fluid and in serum, t1/2 values for cholesterol efflux were in the same range. In addition, estimated maximal efflux values were not significantly different in follicular fluid and serum (45.9% and 49.6%, respectively), as were K(m) values (0.064 and 0.071 mmol/L HDL cholesterol respectively). In addition, isolated HDLs displayed the same capacity to promote cellular cholesterol efflux in both media. Thus, the kinetics and dose-response data between these two physiological media showed that HDLs play the major role in cellular cholesterol efflux. The rate of cholesterol esterification, as measured in the presence of cells, was significantly higher in follicular fluid than in serum at constant HDL cholesterol concentrations, whereas the rate of esterified cholesterol transfer toward added LDL was lower. In contrast, in a cell-free system, lecithin:cholesterol acyltransferase activity represented only 26% of that in serum HDL, whereas cholesterol ester transfer protein activities were comparable. In summary, in this particular model, we confirmed the essential role of HDLs as physiological acceptors in the removal of cellular cholesterol.

Animals↗
PubMed1997

Evolution of mammalian apolipoprotein A-I and conservation of antigenicity: correlation with primary and secondary structure.

We have evaluated the immunoreactivity of 20 monoclonal antibodies (mAbs) directed against human apolipoprotein (apo)A-I with a panel of high density lipoproteins (HDL) from 13 mammalian species. The pattern of cross-reactivity showed that 20 mAbs had different specificity. While not all mAbs recognized apoA-I from all of the different species, the antigenicity of some sequences was well conserved. Thus, mAb A05 cross-reacted with all species except guinea pig and rat. In contrast, the mAb 4H1, which recognized residues 2-8, required a specific proline in position 3, as no immunoreactivity was found in the species missing this amino acid. Furthermore, the presence of a threonine residue in place of serine (in position 6) in the cynomolgus monkey was associated with a 20-fold loss of immunoreactivity in radioimmunometric assay with 4H1. As most of the epitopes were found in CNBr fragments 2 and 3, we sequenced these regions in four species (horse, goat, sheep, and cat) and analyzed the alignment of most known sequences to evaluate their consensus. Except for the rat and the chicken, considerable identity was observed. This permitted us to deduce the involvement of the residues in some antigenic epitopes. In the middle of apoA-I, a conservative mutation Asp103-->Glu was found sufficient to eliminate all reactivity of this epitope for A11 (residues 99-105 ... 12l6-132) in five species (rabbit, cow, goat, sheep, and rat). The residues essential to the expression of two other epitopes overlapping with A11 were also characterized. Edmundson-wheel representation of 18-residue repeated sequences of the different apoA-I species (for the eight amphipatic helices of residues 46-63, 68-85, 101-118, 123-140, 143-160, 167-184, 189-206, and 222-239) showed that secondary structure of apoA-I was more conserved than the antigenic epitopes. The N-terminal region, residues 1 to about 98, is rich in both strictly preserved sequences and epitope expression in most of the species surveyed. This evolutionary conservation of the N-terminal domain suggests an important yet unknown function.

Amino Acid Sequence↗
PubMed1996

High-density lipoprotein 3 receptor-dependent endocytosis pathway in a human hepatoma cell line (HepG2).

The internalization of HLD3 into HepG2 cells at 37 degrees C was precisely measured, taking advantage of the previously observed rapid dissociation of HDL3 from its two binding sites [Barbaras, R., et al. (1994) Biochemistry 33, 2335-2340]. We observed a high level of HDL3 internalization (100 ng/mg of cell protein, corresponding to 45.5% of the total HDL3 associated to the cells at 37 degrees C) reaching a plateau at 15 min. Apolipoprotein A-I (the main HDL3 apolipoprotein) associated with dimyristoylphosphatidylcholine (DMPC) complexes was also internalized by HepG2 cells, at levels comparable to those obtained with HDL3 lipid-free apolipoprotein A-I, which can bind only to the HDL3 high-affinity binding site, and displayed a weak internalization (5 ng internalized/mg of cell protein compared to 250 ng/mg for apolipoprotein A-I complexed with DMPC). Clathrin-coated vesicle purification following HDL3 or LDL internalization at 37 degrees C showed radioactivity associated with these vesicles, and further content analysis evidenced the presence of radiolabeled apoA-I and apoB, respectively. Treatment of the cells either by saccharose hypertonic shock or by potassium depletion, in order to block clathrin-coated vesicle formation, completely inhibited HDL3 internalization, as also observed with LDL. Altogether, these observations clearly demonstrate that HDL3 internalization into HepG2 cells occurs through an endocytosis pathway involving an interaction between apolipoprotein A-I and a cell surface protein, leading to the formation of clathrin-coated vesicles.

Apolipoprotein A-I↗
PubMed1996

Biochemical characterization of pre-beta 1 high-density lipoprotein from human ovarian follicular fluid: evidence for the presence of a lipid core.

In order to isolate pre-beta 1 HDL, we have focused our interest on a particular model, namely, human preovulatory follicular fluid, which contains only HDL as a lipoprotein class as well as a high proportion of pre-beta 1 HDL relative to total HDL (1.5 times more than in homologous plasma) as evidenced by double-dimension gel electrophoresis. Apo A-I in pre-beta 1 HDL represented 17.6% of total apo A-I. Stokes' radii corresponded to 3.42 nm in follicular fluid pre-beta 1 HDL and 3.48 nm in homologous plasma counterparts. After electroelution from agarose, pre-beta 1 HDL were isolated in amounts sufficient to allow characterization by size-exclusion chromatography using HPLC. The estimated apparent molecular mass of these particles is 61.6 kDa. Lipid composition of pre-beta 1 HDL evidenced a low lipid content compared to follicular fluid HDL isolated by ultracentrifugation. Phospholipid composition showed a dramatic decrease in phosphatidylcholines (40.5% of total phospholipids), and the presence of lysophosphatidylcholines and of acidic phospholipids such as phosphatidylserine and phosphatidylinositol (13.6 and 13.7%, respectively). Furthermore, cholesteryl ester and triacylglycerol molecules were quantified by gas-liquid chromatography and represented 8-9% of the pre-beta 1 HDL total weight. Thus, a lipid core is present in pre-beta 1 HDL, which would be compatible with a spherical shape. The follicular fluid appears to be a good model to a better understanding of HDL metabolism.

Apolipoprotein A-I↗
PubMed1996

Identification and quantification of diacylglycerols in HDL and accessibility to lipase.

We have investigated the presence of diacylglycerols in lipoproteins and especially in HDL. Lipoprotein diacylglycerols are very difficult to isolate and to quantify using classical enzymatic techniques, as they are measured in the presence of triacylglycerols and monoacylglycerols. Using a rapid and very sensitive method of gas-liquid chromatography, developed for neutral lipid analysis on an Ultra 1 Hewlett-Packard fused silica capillary column, diacylglycerols (DG) were identified in HDL and classified into five groups: DG 14-16, DG 16-16, DG 16-18, DG 18-18, and DG 18-20. However, their quantitation was difficult due to only partial resolution of molecular species. HDL lipids were submitted to preparative gas-liquid chromatography and diacylglycerols were then silylated using trimethylsilyl reagents. The trimethylsilyl ethers were analyzed by gas-liquid chromatography on a Restek 50 capillary column and were resolved on the basis of carbon number, degree of unsaturation, and double bond positions. The amount of HDL diacylglycerols was twice that of triacylglycerols. The major molecular species of diacylglycerols consisted of 16:0-18:2n-6, 18:0-18:2n-6, and 16:0-18:1n-9 as the major molecular species (33.4, 22.2, and 16.1 mol % of total diacylglycerols, respectively). Using guinea pig cationic pancreatic lipase in order to test the accessibility of diacylglycerols at the surface of HDL, we measured 59% of diacylglycerol hydrolysis, whereas no triacylglycerol hydrolysis was obtained. In addition, most of diacylglycerols having long chain fatty acids, such as 18-20, were completely hydrolyzed, whereas 18-18 and 16-18 were only partially hydrolyzed (64 and 46% respectively). This reflects a different partition of diacylglycerol molecular species between the particle's surface and the lipid core in HDL. This is the first analysis of diacylglycerol molecular species and their distribution in native lipoprotein particles.

Chromatography, Gas↗
PubMed1995

Distribution, fatty acid composition and apolipoprotein A-I immunoreactivity of high density lipoprotein subfractions in myocardial infarction.

The lipoprotein profile, the HDL subfractions and the apolipoprotein (apo) A-I conformation of HDL were determined in 119 patients with myocardial infarction and 119 controls, paired for age. Apo A-I conformation was assessed by its immunoreactivity towards two monoclonal antibodies, 5F6 and 3G10. HDL phospholipid levels and fatty acid composition were also determined. HDL and HDL3 cholesterol, apo A-I, LpA-I and LpA-I:A-II levels were significantly lower in patients than in controls, whereas the levels of total and LDL-cholesterol and of apo B were not different. Apo A-I immunoreactivity against MAb 5F6 was significantly better in patients than in controls, while the immunoreactivity against 3G10 was similar. Paired stepwise logistic regression showed only apo A-I, HDL3 cholesterol and 5F6 immunoreactivity to be significantly related to myocardial infarction. After adjustment for HDL cholesterol, lower levels of HDL phospholipids and polyunsaturated fatty acids (PUFA) were found in patients compared with controls. Hence, of all HDL markers, apo A-I and HDL3 cholesterol appear to be the most informative. Also, lower HDL phospholipid and PUFA content may explain a different apo A-I conformation in patients with myocardial infarction.

Adult↗
PubMed1995

Relationships of plasminogen activator inhibitor activity and lipoprotein(a) with insulin, testosterone, 17 beta-estradiol, and testosterone binding globulin in myocardial infarction patients and healthy controls.

The relationships between plasminogen activator inhibitor (PAi) activity and lipoprotein(a) [Lp(a)] and insulin, testosterone, 17 beta-estradiol, and testosterone binding globulin (TEBG) were assessed in 42 myocardial infarction male patients and 74 healthy controls. Patients had higher levels of insulin than did controls (87 +/- 30 vs. 75 +/- 28 pmol/L, respectively; P < 0.04), and no differences were found in levels of PAi activity, testosterone, 17 beta-estradiol, and TEBG. Lp(a) levels greater than 0.3 g/L were more frequent in patients than in controls (P < 0.002). In all subjects, PAi activity levels were significantly and positively correlated with body mass index (r = 0.20, P < 0.05), triglycerides (r = 0.38, P < 0.0001), and insulin (r = 0.27, P < 0.005) and were negatively correlated with testosterone (r = -0.28, P < 0.005) and TEBG (r = -0.42, P < 0.001). Stepwise multiple regression analysis showed triglyceride, insulin, and TEBG levels to be significantly related to PAi activity. No significant correlations were found between Lp(a) levels and all hormonal variables studied and between Lp(a) and PAi activity (r = -0.06, P < 0.58). These results suggest that TEBG is significantly and independently related to PAi levels.

Adult↗
PubMed1994

Hepatic lipase induces the formation of pre-beta 1 high density lipoprotein (HDL) from triacylglycerol-rich HDL2. A study comparing liver perfusion to in vitro incubation with lipases.

High density lipoprotein subfractions with a pre-beta migration play a key role in the reverse cholesterol transport. The origin of these particles is not yet clearly defined. We propose to verify a possible origin of these particles during the catabolism of high density lipoprotein2 (HDL2) by hepatic lipase using two different models. A rat liver perfusion of native human HDL2 in the presence of heparin induced, after 30 min, the formation of the pre-beta 1 HDL subspecies. Human HDL2 enriched with triacylglycerols, perfused in the same conditions, led after 15 min to an enhanced production of this pre-beta 1 HDL population, as compared with the results obtained with native HDL2. A reduction of the alpha-HDL2 fraction was also evident. After perfusion, a similar formation of pre-beta 1 HDL from triacylglycerol-rich HDL2 was observed in absence of heparin. When these HDL2 were incubated in vitro for 120 min at 37 degrees C in the presence of partially purified rat hepatic lipase, the appearance of pre-beta 1 HDL was again found and associated with a decrease in size of the remaining alpha-HDL subfractions as compared with original HDL2. On the contrary, the incubation of the same HDL2 with snake venom phospholipase A2 produced no pre-beta HDL. These results evidence the role of the triacylglycerol lipase activity of hepatic lipase in the formation of pre-beta 1 HDL from triacylglycerol-rich HDL2.

Animals↗
PubMed1994

Specific binding of free apolipoprotein A-I to a high-affinity binding site on HepG2 cells: characterization of two high-density lipoprotein sites.

In this paper, we present the first evidence that free apoA-I, without association with lipids, binds only to a high-affinity binding site (Kd = 1.8 microgram/mL, Bmax = 63.12 ng/mL). This is a new binding site of higher affinity (80-100 times) but of lower capacity than the binding sites already described for HDL. This is also the first evidence on HepG2 cells both of a high-affinity site (Kd = 0.685 microgram/mL, Bmax = 39.86 ng/mL) and of a low-affinity site (Kd = 55.65 micrograms/mL, Bmax = 665.45 ng/mL) for HDL. ApoA-I-DMPC complexes also present two binding components comparable to the HDL3 binding sites. This free apoA-I binding is specific, as shown by competition experiments, and allowed us to specifically study this high-affinity site, without interference of the low-affinity one. Kinetic rates of association/dissociation for the high-affinity site were faster than for the low-affinity site (10 and 20 min versus 40 and 30 min, respectively). The kinetic Kd values, derived from association and dissociation rate constants (Kd = 55.14 and 2.91 micrograms/mL), were of similar magnitude as the Kd values calculated by Scatchard analysis. These data confirm that HDL3 binding sites characterized by saturation experiments follow the law of mass action, indicative of ligand-receptor interaction. In summary, HepG2 cells present high HDL3 binding sites which are able to bind free apoA-I in contrast with the low-affinity HDL3 binding sites.

Apolipoprotein A-I↗
PubMed1994

Hepatic lipase promotes the uptake of HDL esterified cholesterol by the perfused rat liver: a study using reconstituted HDL particles of defined phospholipid composition.

The role of hepatic triacylglycerol lipase (H-TGL) in promoting the liver uptake of high density lipoprotein (HDL) free and esterified cholesterol was studied in a recirculating rat liver perfusion, a situation where the enzyme is physiologically expressed and is active at the vascular bed. For this purpose, reconstituted HDL of defined phospholipid composition were prepared, containing either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, a substrate for H-TGL, or 1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphocholine, a non-hydrolyzable analog. Reconstituted HDL were then used in the perfused rat liver system. The main results are the following. 1) Reconstituted HDL were obtained by sonication of lipids and apolipoproteins and isolated by ultracentrifugation in the 1.07-1.21 g/ml density interval. Reconstituted HDL containing either diacylphosphatidylcholine or alkyl-acyl-phosphatidylcholine were similar in terms of chemical composition, apparent size, and apolipoprotein A-I immunoreactivity, and were comparable to native HDL3. 2) Reconstituted HDL were labeled with free [14C]cholesterol and [3H]cholesteryl ether, a non-hydrolyzable tracer of esterified cholesterol, and were perfused through the rat liver. Liver uptake of [3H]cholesteryl ether was 2.5-fold higher from reconstituted HDL containing diacylphospholipid than from HDL reconstituted with alkyl-acyl-phospholipids. Liver uptake of free [14C]cholesterol was identical in both cases. 3) H-TGL-depleted rat livers were obtained by a 12-min preperfusion in the presence of heparin, displacing 90% of the enzymatic activity. The residual activity in the perfusate was inhibited by a specific antibody directed against rat H-TGL. Liver uptake of [3H]cholesteryl ether from reconstituted HDL containing diacylphospholipid was reduced by 35% in hepatic lipase-depleted livers compared to controls. On the other hand, hepatic lipase depletion had no effect on the liver uptake of esterified cholesterol from HDL reconstituted with alkyl-acyl-phospholipids. The above findings support a role for the phospholipase A1 activity of H-TGL in stimulating the delivery of HDL esterified cholesterol to liver cells.

Animals↗