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Biomedical subjects

L Calabresi

Publications and source records attributed to L Calabresi.

At least 37 records · Page 2Linked to original sources
PubMed1997

Inhibition of VCAM-1 expression in endothelial cells by reconstituted high density lipoproteins.

Plasma-derived high density lipoproteins (HDL) were found to inhibit cytokine-induced expression of endothelial cell adhesion molecules. Here we used apolipoprotein-specific reconstituted HDL (rHDL) made with phosphatidylcholine (PC) and three different apolipoproteins to identify the HDL components involved in this effect. rHDL containing apolipoprotein A-I (apoA-I), the disulfide-linked form of the apoA-IMilano variant, or apoA-II, were all effective in inhibiting the expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 in TNF alpha- or LPS-stimulated HUVEC. The inhibition was concentration dependent in the range of 0.1-1.0 mg/ml (protein). PC liposomes slightly depressed TNF alpha-induced VCAM-1 expression (16% vs 43-50% for the various rHDL), whereas the lipid-free apolipoproteins had no effect. The protein component of HDL is involved in the inhibition of VCAM-1 expression in HUVEC through a rather unspecific mechanism, as three apolipoproteins with remarkably different primary structure display very similar activity.

Apolipoprotein A-I↗
PubMed1997

Denaturation and self-association of apolipoprotein A-I investigated by electrophoretic techniques.

Purified human apolipoprotein A-I (apoA-I), when run across a transverse urea gradient at alkaline pH, gives a complex pattern characterized by a number of parallel sigmoidal curves, in which the transition between high- and low-mobility forms, i.e. from folded to unfolded structure, occurs between 1.1 and 3.2 M urea. Size differences appear to be the major cause of this isomerism. When migrated across a wide pH range in the presence of varying amounts of urea to display its titration curve, apoA-I is resolved into two pairs of bands, running parallel in the neutral to basic pH region while merging at acidic pH; such a finding does not correlate with a differential exposure of His residues, as shown by diethyl pyrocarbonate titration. Ferguson plot analysis, confirmed by cross-linking experiments, demonstrates a gradual shift from higher to lower mass aggregates as the urea concentration is raised; the monomeric form undergoes denaturation by swelling to an approximately 50% larger hydrodynamic volume than in its native state. At alkaline pH, where apoA-I exists as aggregated species, disaggregation and unfolding appear to happen at once, the larger aggregates being less stable than the smaller ones. At acidic pH, apoA-I does not form aggregates and has little secondary structure; unfolding is then a progressive rather than a cooperative process.

Apolipoprotein A-I↗
PubMed1997

Activation of lecithin cholesterol acyltransferase by a disulfide-linked apolipoprotein A-I dimer.

Apolipoprotein A-IMilano is a molecular variant of apoA-I, containing the Arg173-->Cys substitution that forms a disulfide linked homodimer (A-IM/A-IM). To assess the effect of this structural modification on a major function of the apolipoprotein, its activation of lecithin cholesterol acyltransferase (LCAT), we prepared well-defined complexes of A-IM/A-IM and apoA-I with phospholipids and cholesterol and compared their reactivities with LCAT. Particles with A-IM/A-IM had very similar diameters to apoA-I particles (7.8 and 12.5 nm) but had distinct apolipoprotein and phospholipid contents and protein secondary structures; they bound LCAT with comparable affinities, but were less efficient substrates for the enzyme (40 to 70% less reactive). We conclude that the local structural constraints in A-IM/A-IM do not prevent the formation of well-defined complexes with phospholipids and do not influence the binding of the enzyme to the particles, but have an inhibitory effect on LCAT activation.

Apolipoprotein A-I↗
PubMed1997

High density lipoprotein and coronary heart disease: insights from mutations leading to low high density lipoprotein.

Hypoalphalipoproteinemia can result from defects in the genes encoding apolipoprotein A-I, the major protein component of HDL, or enzymes that are critical for the formation/maturation of mature HDL. Recent information contradicts earlier findings, suggesting that most of the affected subjects are at increased risk of developing coronary heart disease, independent of the mutated gene. A possible exception is represented by mutations in the apolipoprotein A-I gene leading to structural variants, that might even exert a protective effect against atherosclerosis.

Apolipoprotein A-I↗
PubMed1996

Human apolipoprotein A-II inhibits the formation of pre-beta high density lipoproteins.

The role of human apolipoprotein A-II (apoA-II) in the remodeling of human high density lipoproteins (HDL) was investigated during incubation of native and reduced-carboxamidomethylated (RCM) HDL3 with a lipoprotein-depleted plasma fraction (LPDP) in the presence of triglyceride-rich particles (TGRP) isolated from Intralipid. Reduction-carboxamidomethylation of HDL3 entirely converts the disulfide-linked apoA-II dimers into monomers, without affecting the structure, composition and particle size distribution of HDL3. Following incubation with LPDP and TGRP, unmodified HDL3 are mainly converted into large, HDL2 particles (diameter: 9.90 +/- 0.07 nm), enriched in triglycerides and depleted of cholesteryl esters. RCM-HDL3 are converted into both large HDL2 (9.86 +/- 0.07 nm) and small (7.53 +/- 0.06 nm) HDL3. The small products are protein-rich and cholesterol-poor, and consist of two different particles: a component with pre-beta mobility, containing only apoA-I, and a component with alpha mobility, containing both apoA-I and apoA-II. Kinetic studies suggest that a two-step process is involved in the formation of small, pre beta-HDL3, by which changes in lipid composition cause alterations in lipoprotein structure/stability, favoring the dissociation of apolipoproteins and reduction of particle size. These findings indicate that apolipoprotein structure is a major determinant of HDL remodeling, apoA-II potentially counteracting the anti-atherogenic properties of apoA-I by inhibiting the formation of small, pre-beta-migrating HDL.

Adult↗
PubMed1996

The molecular structure of apolipoprotein A-II modulates the capacity of HDL to promote cell cholesterol efflux.

The influence of apolipoprotein A-II (apoA-II) molecular structure on the capacity of high density lipoproteins (HDL) to promote cellular cholesterol efflux was investigated in cultured mouse peritoneal macrophages (MPM). Conversion by reduction and carboxamidomethylation of the naturally occurring dimeric apoA-II to its monomeric form in both native or reconstituted HDL did not change apolipoprotein secondary structure and lipoprotein size/composition. All particles containing monomeric apoA-II, i.e., native HDL3 or reconstituted HDL with or without apoA-I, showed a higher ability to promote cholesterol efflux originating from plasma membrane and intracellular stores, compared to particles containing dimeric apoA-II. These findings indicate that apolipoprotein molecular structure is a major determinant of HDL capacity to promote cholesterol efflux from cells.

Animals↗
PubMed1995

Gene dose of the epsilon 4 allele of apolipoprotein E and disease progression in sporadic late-onset Alzheimer's disease.

A strong association has been described in cross-sectional studies between the epsilon 4 allele of the apolipoprotein E and late-onset Alzheimer's disease (LOAD). This study addresses the relationship between disease progression and the epsilon 4 allele in 62 sporadic LOAD (onset at age 70 and over) patients. Disease progression was estimated retrospectively with the Mini-Mental State Examination (MMSE) amounting to 4.7, 3.8, and 2.2 points per year (sex-adjusted test for trend: p = 0.01) and with the Clinical Dementia Rating (CDR) to 0.76, 0.67, and 0.42 units per year (p = 0.03) in -/-, epsilon 4/-, and epsilon 4/epsilon 4 patients. The proportion of patients with fast progression decreased (p < or = 0.005) and with slow progression increased (p = 0.002) with increasing epsilon 4 gene dose. These findings were confirmed in a smaller sample of 28 patients for whom longitudinal assessments of MMSE were available and when nonlinear progression of the disease was accounted for in a stratified analysis. These data suggest that disease duration might be longer in epsilon 4 carriers and that this might at least partly account for the cross-sectional association between the epsilon 4 allele and LOAD.

Age of Onset↗
PubMed1995

Triglycerides are major determinants of cholesterol esterification/transfer and HDL remodeling in human plasma.

Lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) are responsible for the esterification of cell-derived cholesterol and for the transfer of newly synthesized cholesteryl esters (CE) from HDL to apoB-containing lipoproteins in human plasma. LCAT and CETP are also crucial factors in HDL remodeling, a process by which HDL particles with a high capacity for cell cholesterol uptake are generated in plasma. In the present study, cholesterol esterification and transfer were evaluated in 60 patients with isolated hypercholesterolemia (HC, n = 20) and isolated (HTG, n = 20) or mixed hypertriglyceridemia (MHTG, n = 20) and in 20 normolipidemic healthy individuals (NL). Cholesterol esterification rate (CER) and net CE transfer rate (CETR) were measured in whole plasma. LCAT and CETP concentrations were determined by specific immunoassays. HDL remodeling was analyzed by monitoring changes in HDL particle size distribution during incubation of whole plasma at 37 degrees C. Mean CER and CETR were 48% and 73% higher, respectively, in hypertriglyceridemic (HTG + MHTG) versus normotriglyceridemic individuals. HDL remodeling was also significantly accelerated in plasma from hypertriglyceridemic patients. Strong positive correlations were found in the total sample between plasma and VLDL triglyceride levels and CER (r = .722 and r = .642, respectively), CETR (r = .510 and r = .491, respectively), and HDL remodeling (r = .625 and r = .620, respectively). No differences in plasma LCAT and CETP concentrations were found among the various groups except for a tendency toward higher CETP levels in hypercholesterolemic patients (+51% in MHTG and +20% in HC) versus control subjects (NL). By stepwise regression analysis, VLDL triglyceride level was the sole significant predictor of CER and CETR and contributed significantly together with baseline HDL particle distribution to HDL remodeling. These results indicate that plasma triglyceride level is a major factor in the regulation of cholesterol esterification/transfer and HDL remodeling in human plasma, whereas LCAT/CETP concentrations play a minor role in the modulation of reverse cholesterol transport.

Carrier Proteins↗
PubMed1994

Molecular characterization of native and recombinant apolipoprotein A-IMilano dimer. The introduction of an interchain disulfide bridge remarkably alters the physicochemical properties of apolipoprotein A-I.

The disulfide-linked dimer of apolipoprotein A-IMilano (A-IM/A-IM), a natural Arg173-->Cys variant of apoA-I, was purified from carriers' plasma and produced in Escherichia coli. The recombinant A-IM/A-IM is identical to native A-IM/A-IM, by mass spectrometry, SDS-polyacrylamide gel electrophoresis, and isoelectric focusing. Lipid-free A-IM/A-IM undergoes concentration-dependent self-association similar to apoA-I, but at all concentrations apoA-I is more self-associated than A-IM/A-IM. Far-ultraviolet CD spectra of A-IM/A-IM reveal a highly alpha-helical structure predicted to be approximately 65% in the lipid-free and approximately 78% in the lipid-associated states, versus 43 and 73% for apoA-I. A significant loss of alpha-helix occurs below pH 3.5 and above pH 10 in both apoA-I and A-IM/A-IM; A-IM/A-IM constantly shows a higher alpha-helical content than apoA-I over the entire pH range (1.7-12.8), suggesting that hydrophobic forces stabilize the interaction between the two A-IM chains. Indeed, and differently from apoA-I, the alpha-helical content of A-IM/A-IM is minimally affected by solvent ionic strength. The aromatic side chains in both lipid-free and lipid-bound A-IM/A-IM are immobilized in a more asymmetric and hydrophobic environment than in lipid-free apoA-I, the conformation of A-IM/A-IM being instead similar to that achieved by apoA-I following interaction with lipids. The present findings prove that rA-IM/A-IM is structurally identical to the native protein; the conformation of A-IM/A-IM is remarkably different from that of apoA-I, thus possibly explaining some of the peculiar functional properties of the apoA-IMilano dimer.

Amino Acid Sequence↗
PubMed1994

Drug control of reverse cholesterol transport.

Reverse cholesterol transport identifies a series of metabolic events resulting in the transport of excess cholesterol from peripheral tissues to the liver. High-density lipoproteins (HDL) are the vehicle of cholesterol in this reverse transport, a function believed to explain the inverse correlation between plasma HDL levels and atherosclerosis. An attempt to stimulate, by the use of drugs, this transport process may hold promise in the prevention and treatment of arterial disease. Among the agents affecting lipoprotein metabolism, only probucol exerts significant effects on reverse cholesterol transport, by stimulating the activity of the cholesteryl ester transfer protein and, consequently, altering HDL subfraction composition/distribution. Another approach to the stimulation of reverse cholesterol transport consists of raising plasma HDL levels; studies in animals, either by exogenous supplementation or by endogenous overexpression, have shown a consistent benefit in terms of atherosclerosis regression and/or non-progression. Thus, it is time to consider different future treatments of atherosclerosis, combining the classical lipid-lowering treatments with innovative methods to promote cholesterol removal from the arterial wall.

Animals↗
PubMed1993

Apolipoprotein A-I domains involved in the activation of lecithin:cholesterol acyltransferase. Importance of the central domain.

The reaction of highly purified lecithin:cholesterol acyltransferase (LCAT) with defined reconstituted discoidal apoA-I-containing lipoproteins (LpA-I) with 2, 3, or 4 apoA-I molecules/particle (Lp2, 3, or 4A-I) has been studied in the presence of a number of specific anti apoA-I antibodies. Among nine anti-apoA-I monoclonal antibodies (mAbs) reacting with epitopes distributed over 80% of the sequence, three significantly inhibit the LCAT reaction with all particles. The position of their epitopes located in the middle to COOH-terminal region between residues 96-121 (3G10), 135-148 (A03), and 149-186 (A44) is compatible with an inhibition by steric hindrance over a central domain. Antibody 4H1 binding to the NH2 terminus (residues 2-8) profoundly increases (5-fold) the LCAT reaction with Lp2A-I (7.8 nm), but not with other particles. Other mAbs, A11 and 5F6, binding to epitopes (residues 99-139 and 118-141) enhance LCAT reactivity with the small Lp2A-I (7.8 nm) and Lp3A-I (10.8 nm) but not with their larger counterparts. Most mAbs have similar effects on LCAT reaction with native high density lipoprotein3 as with LpA-I. The inhibitory or enhancing effects of these mAbs are also observed with Fab fragments and not related to their binding affinity for apoA-I containing reconstituted lipoprotein particles. The intercalation of epitopes for mAbs that inhibit or enhance LCAT reaction with small LpA-I is compatible not with steric hindrance but with conformational modifications of apoA-I and indirectly of the lipids in small particles. We propose that enhancing mAbs act by stabilization of an apoA-I conformation which is not favored in small LpA-I, i.e. by increasing binding of amphipathic helices to lipids or by interfering with the mobility of a hinged domain. The epitopes for the inhibitory mAbs can be shown to overlap on several LpA-I models, indicating that steric hindrance over a single site is a possible mechanism of inhibition.

Antibodies, Monoclonal↗
PubMed1993

Apolipoprotein A-I conformation in discoidal particles: evidence for alternate structures.

To define the conformation of apolipoprotein A-I in discoidal particles, the immunoreactivity of a series of epitopes distributed along the apoA-I sequence has been evaluated in lipid-free apoA-I and in lipid-bound form. To this end, reconstituted discoidal lipoproteins, here called LpA-I, and defined by number of apoA-I per particle (e.g., Lp2A-I), have been prepared with palmitoyloleoylphosphatidylcholine, cholesterol, and apoA-I. Four LpA-I have been obtained and studied: two in the Lp2A-I class, 7.8 and 9.6 nm in diameter, and two in the Lp3A-I class, 10.8 and 13.4 nm. The immunoreactivity of all the epitopes tested was significantly different in LpA-I particles compared to lipid-free apoA-I, demonstrating that binding to lipids produces a drastic change in apoA-I conformation. Specific domains in the primary sequence become highly exposed while others are masked. Although the variation in immunoreactivity of the epitopes between various LpA-I was not drastic, significant differences in the calculated ED50 values were observed for a number of antibodies in small versus large particles within each class (Lp2A-I or Lp3A-I), indicating that particle size can modulate apoA-I conformation. In addition, when the competition between pairs of mAbs was analyzed in order to understand the relative position of epitopes, highly significant differences were observed as a function of particle size within each class. In particular, the competition between mAbs recognizing epitopes in the central region of apoA-I was greater in the larger particles than in their small counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1993

Increased postprandial lipemia in Apo A-IMilano carriers.

Plasma lipid/lipoprotein changes were monitored after a fat load (65 g fat per square meter body surface area) in six carriers of the apolipoprotein A-IMilano (A-IM) variant and six age- and sex-matched control subjects. The magnitude of postprandial lipemia, calculated as the area under the curve (AUC) described by plasma triglyceride (TG) level versus time, was threefold higher in the A-IM carriers; however, after correction for the different baseline TG levels, it was similar to control subjects. Moreover, the magnitude of postprandial lipemia was positively correlated with baseline TG in both A-IM carriers (r = 0.77) and control subjects (r = 0.80), indicating that fasting TGs are a major determinant of postprandial response in all subjects. Postprandial lipemia was also inversely correlated with high density lipoprotein (HDL) and HDL2 cholesterol in both groups (A-IM, r = -0.81 and -0.79; control subjects, r = -0.87 and -0.94). Different from those in control subjects, the plasma apo A-I levels in the A-IM carriers decreased progressively while apo B increased up to 4 hours but decreased thereafter. Postprandial rises of low density lipoprotein TG but not of HDL-TG AUC were significantly higher in the A-IM carriers, even after normalization for the different fasting concentrations. These data show that the low plasma HDL levels of A-IM carriers, which are secondary to a primary structural alteration of the major HDL apolipoprotein, are associated with elevated fasting and postprandial TG levels and an anomalous postprandial redistribution of TG among lipoprotein classes.

Adult↗
PubMed1992

Apolipoprotein A-II modulates HDL remodeling in plasma.

Native and reduced-carboxamidomethylated (RCM) HDL3 are incubated with a lipoprotein-depleted plasma fraction in the presence of triacylglycerol-rich particles isolated from Intralipid. Unmodified HDL3 are mainly converted into large HDL2b particles (diameter: 9.84 +/- 0.15 nm); RCM-HDL3 are transformed into both large HDL2b (9.76 +/- 0.10 nm) and small HDL3c (7.68 +/- 0.07 nm). These findings indicate that the apo A-II dimers participate in the remodeling of HDL, by inhibiting the formation of small HDL particles.

Animals↗
PubMed1992

Activity profile of gemfibrozil on the major plasma lipoprotein parameters.

Gemfibrozil (G) is a widely used and highly effective fibric acid derivative. It acts both on lipoprotein disorders characterized by hypertriglyceridemia, as well as in hyperchlolesterolemia. The results of two separate studies are summarized, in order to elucidate the differential activity profile of the drug on the major plasma lipoprotein parameters. In a group of hypertriglyceridemic patients (Study I) the major activity of G was on the VLDL cholesterol (C) and triglyceride levels (respectively -40.4 and -35.7%), associated with a marked increase of the HDL-C levels, in particular in the HDL3 subfraction. In addition, apolipoprotein B levels were significantly reduced (-12%) in all treated patients. In Study II, treatment with G in severe hypercholesterolemia was compared to that with pravastatin, a major hydroxymethylglutaryl coenzyme A reductase inhibitor. G, in these patients, markedly reduced both total (-21.6%) and LDL-cholesterol (-24.4%) levels. The increase of HDL-C was less marked than in type IV patients but G, similarly to pravastatin, induced a significant reduction of apolipoprotein B, together with an increase (+19.2%) of apolipoprotein AII levels. These findings indicate that the activity profile of G is wider than expected and that it may include a reduction of LDL levels in severely hypercholesterolemic patients. The mechanism of this latter change is difficult to establish, although it may also be related to an improved delipidation of LDL, resulting in a more efficient interaction with peripheral high affinity receptors.

Adult↗
PubMed1991

Omega-3 fatty acids selectively raise high-density lipoprotein 2 levels in healthy volunteers.

The effects of omega-3 fatty acid supplementation on high-density lipoprotein (HDL) subfraction distribution and composition were evaluated in five healthy volunteers taking 2.8 g/d of eicosapentaenoic acid (EPA) and 1.7 g/d of docosahexaenoic acid (DHA) for 6 weeks. This supplementation resulted in marked changes of the plasma fatty acid composition. Plasma total cholesterol (TC), HDL-cholesterol (HDL-C), and triglyceride (TG) levels did not change. HDL2-C increased by 74%, with a concomitant 19% decrease of HDL3-C; the HDL2 to HDL3 mass ratio increased from 0.30 +/- 0.19 to 0.47 +/- 0.28. The increase of HDL2 was confirmed by nondenaturing polyacrylamide gradient gel electrophoretic separation of HDL subclasses, otherwise showing no change in HDL particle size. After omega-3 supplementation, both HDL2 and HDL3 became cholesteryl ester (CE)- and TG-enriched and free cholesterol (FC)- and phospholipid (PL)-depleted. The reported findings provide a useful adjunct to the antithrombotic potential of omega-3 fatty acids.

Adult↗