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F Nigon

Publications and source records attributed to F Nigon.

At least 19 recordsLinked to original sources
PubMed2005

DNMT3B mutations and DNA methylation defect define two types of ICF syndrome.

ICF syndrome is a rare autosomal recessive disease characterized by variable immunodeficiency, centromeric instability, and facial abnormalities. Mutations in the catalytic domain of DNMT3B, a gene encoding a de novo DNA methyltransferase, have been recognized in a subset of patients. ICF syndrome is a genetic disease directly related to a genomic methylation defect that mainly affects classical satellites 2 and 3, both components of constitutive heterochromatin. The variable incidence of DNMT3B mutations and the differential methylation defect of alpha satellites allow the identification of two types of patients, both showing an undermethylation of classical satellite DNA. This classification illustrates the specificity of the methylation process and raises questions about the genetic heterogeneity of the ICF syndrome.

Centromere↗
PubMed2001

Characterization of human apolipoprotein B100 oligosaccharides in LDL subfractions derived from normal and hyperlipidemic plasma: deficiency of alpha-N-acetylneuraminyllactosyl-ceramide in light and small dense LDL particles.

The carbohydrate composition of apolipoprotein (apo) B100, particularly its degree of sialylation, may contribute to the atherogenic properties of low-density lipoprotein (LDL). We analyzed LDL apoB100 glycans derived from normolipidemic, hypercholesterolemic, and hypertriglyceridemic diabetic subjects. Using exoglycosidase carbohydrate sequencing and matrix-assisted laser desorption/ionization mass spectrometry to analyze fluorescently labeled oligosaccharides, we report evidence for several carbohydrates not previously identified on apoB100, including truncated complex biantennary N-glycans and hybrid N-glycans. The distribution and diversity of the apoB100 glycans isolated from all individuals was highly conserved. The N-glycan composition of apoB100 derived from five LDL subpopulations (LDL1, d = 1.018-1.023; LDL2, d = 1.023-1.030; LDL3, d = 1.030-1.040; LDL4, d = 1.040-1.051; LDL5, d = 1.051-1.065 g/ml) did not vary in normolipidemic or hypercholesterolemic subjects. Furthermore, we found no evidence for "desialylated" apoB100 glycans in any of the samples analyzed. Analysis of the most abundant LDL ganglioside, alpha-N-acetylneuraminyllactosyl-ceramide, revealed a deficiency in small dense LDL and in the most buoyant subpopulation. These data provide a novel explanation for the apparent deficiency of sialic acid in small dense LDL and indicate that the global apoB100 N-glycan composition is invariable in the patient groups studied.

Apolipoprotein B-100↗
PubMed2001

Plant sterol-enriched margarine lowers plasma LDL in hyperlipidemic subjects with low cholesterol intake: effect of fibrate treatment.

Phytosterols, found in fat-soluble fractions of plants, chemically resemble cholesterol and inhibit cholesterol absorption in the small intestine. Phytosterol consumption in human subjects reduces plasma total and low density lipoprotein-cholesterol (LDL-C) levels. The primary aim of this study was to determine the efficacy of a low-fat spread enriched with plant sterols in reducing total and LDL-C concentrations in primary hypercholesterolemia. The secondary objective was to evaluate whether patients receiving a lipid-lowering drug (fibrate) might differ in their response to plant sterols. The study was a randomized, double-blind, placebo-controlled two-period cross-over trial with two treatments and three periods. Both treatment periods lasted 2 months, with a washout period (2 months) between them. Spread enriched with plant sterols was compared to non-enriched control spread. Fortified fat spread provided 1.6 g/day of plant sterols derived from edible vegetable oils and fatty acids from sunflower seed oil. The plant sterol content consisted of sitosterol esters (50%), campesterol esters (25%), stigmasterol esters (20%) and 10% of other esters. Data in 53 hypercholesterolemic patients (31 females and 22 males) who completed the study were as follows: patients were 58+/-12 years of age with mean body mass index 23.5+/-2.8 kg/m2 (mean+/-SD). No adverse side-effects of the diet were reported. Plasma total cholesterol and LDL-C concentrations were significantly reduced by 6.4% and 8.8%, respectively, after using the spread enriched in plant sterols, as compared to controls (0.0% and 1.3%, respectively). No effect on high density lipoprotein-cholesterol (HDL-C) and lipoprotein(a) concentrations was detected. When subjects were divided in two subgroups according to fibrate treatment, supplementation with phytosterols decreased plasma cholesterol and LDL-C by 8.5% and 11.1%, respectively in the subgroup of patients treated with fibrates. In the group of patients who did not receive fibrates, consumption of plant sterol margarine reduced plasma cholesterol and LDL-C by 5.5% and 7.7%, respectively. Spread enriched with plant sterol esters significantly lowers blood total and LDL-C levels without affecting HDL-C concentrations, in a hypercholesterolemic population following a strict low cholesterol diet (NCEP step1). In addition, a combination of fibrate treatment and plant sterol ester-supplemented spread offers a safe and effective measure to significantly decrease abnormally high cholesterol levels. We conclude that phytosterol-enriched spread is a useful adjunctive therapy for hypercholesterolemic patients.

Adult↗
PubMed2000

Kinetics of tryptophan oxidation in plasma lipoproteins by myeloperoxidase-generated HOCl.

The relative susceptibility of the apoprotein components of human lipoproteins [high-density lipoprotein (HDL) and low-density lipoprotein (LDL)] and their subclasses to oxidation by the myeloperoxidase/H2O2/Cl- system in vitro was studied by measuring the decrease in rate of tryptophan fluorescence. Whereas the lipoprotein-modification rate showed a saturation type of dependence on the concentration of myeloperoxidase, a biphasic dependence on the concentration of the lipoproteins was found. High concentrations of H2O2 were also found to inhibit tryptophan oxidation in LDL but to a lesser extent in HDL. The optimal rate of LDL and HDL modification was observed at pH 6.0. HDL was modified much more rapidly than LDL, which may be due to differences in size and different relative contents of protein and lipids per particle. No differences in rates of modification of LDL subclasses were observed, when the assays were standardized to equal LDL protein concentrations, but, when standardized to equal particle mass, an optimum at subclass 8 was found, which is probably due to differences in apolipoprotein B-100 conformation. It was concluded that HDL may have a beneficial effect in retarding LDL modification in inflammatory processes.

Humans↗
PubMed2000

Oxidizability of atherogenic low-density lipoprotein subspecies in severe familial hypercholesterolemia: impact of long-term low-density lipoprotein apheresis.

BACKGROUND: Oxidative modification of low-density lipoprotein (LDL) plays a key role in the pathophysiology of atherosclerosis. LDL-apheresis, which involves direct removal of plasma LDL from circulating blood, is an efficient treatment of homozygous familial hypercholesterolemia (FH). METHODS: We evaluated impact of long-term LDL apheresis treatment on the atherogenicity of the major LDL subclasses (light, LDL1, and LDL2, density [d] 1.018-1.030 g/mL; intermediate, LDL3, d 1.030-1.040 g/mL, and dense LDL, LDL4 and LDL5, d 1.040-1.065 g/mL) separated by density gradient ultracentrifugation in severe FH patients. Therefore, we compared the oxidative resistance as well as the chemical and physical properties of each LDL subpopulation in the FH group with those in the corresponding LDL subfractions from normocholesterolemic control subjects. RESULTS: Both intermediate and dense LDL subfractions were significantly more resistant to copper-mediated oxidation in FH patients treated regularly by LDL-apheresis than their counterpart controls. The lag phases for LDL3, LDL4, and LDL5: 63.9+/-11.6, 55.8+/-1.2, and 47.2+/-6.5 min. in FH patients were significantly longer than those of the corresponding subfractions in normocholesterolemic controls (P <.01 for LDL3 and LDL5, P<.005 for LDL4). This protective effect was reflected in the delayed formation of biologically active lipid oxidation products such as oxysterols, lipid hydroperoxides, dienes, and dienals in the intermediate and dense LDL subfractions of FH patients. These findings may result from lower "seed" contents of lipid hydroperoxide (LOOH) detected as dienes in plasma LDL from apheresis-treated FH patients; indeed, baseline LOOH/diene contents in all 5 LDL subclasses from FH patients were significantly lower than those of the corresponding subclasses in normolipidemic subjects (P<.0005). On the other hand, the enhanced oxidative resistance of both intermediate (LDL3) and dense (LDL4 and LDL5) LDL subpopulations in FH patients could not be accounted for by any consistent modification in chemical composition or in lipophilic antioxidant content, although minor differences were observed between patients and controls in unsaturated fatty acid profile. In contrast, sphingomyelin content was enriched in FH LDL subclasses, potentially resulting in reduced penetration of the hydrophilic surface layer of LDL by oxygen radicals. CONCLUSION: We conclude that low concentrations of preformed lipid hydroperoxides and dienes, together with surface sphingomyelin enrichment, can account for the enhanced oxidative resistance of intermediate (LDL3) and atherogenic dense LDL (LDL4, LDL5) induced by long-term LDL apheresis in severe FH patients.

Adolescent↗
PubMed1999

Microphase separation in low density lipoproteins. Evidence for a fluid triglyceride core below the lipid melting transition.

The structural organization of the neutral lipid core in human low density lipoproteins (LDL) was investigated in physicochemically defined, distinct human LDL subspecies in the density range of 1. 0244-1.0435 g/ml by evaluation of the core lipid transition temperature, chemical composition, and the behavior of spin-labeled core lipids. Calorimetric studies were performed on more than 60 LDL preparations, and the transition temperature, which varied between 19 and 32 degreesC, was correlated to the chemical composition and revealed a discontinuity at a critical cholesteryl ester to triglyceride ratio of approximately 7:1. For electron spin resonance studies, several LDL preparations were probed with spin-labeled cholesteryl esters and triglycerides, respectively. In LDL with a high triglyceride content, both labels exhibited similar mobility behavior. In contrast, in LDL with only small concentrations of triglycerides, the behavior of labeled cholesteryl esters and labeled triglycerides differed distinctly. The cholesteryl esters were strongly immobilized below the transition temperature, whereas the triglycerides remained fluid throughout the measured temperatures. These results suggest that the critical cholesteryl ester to triglyceride mass ratio of 7:1 corresponds to two concentric compartments with a radial ratio of 2:1, where the liquid triglycerides occupy the core, and the cholesteryl esters form the frozen shell. At higher triglyceride contents, the triglyceride molecules insert into the cholesteryl ester shell and depress the peak transition temperature of the LDL core, whereas at lower triglyceride contents, excess cholesteryl esters are dissolved in the core.

Calorimetry, Differential Scanning↗
PubMed1999

Cholesteryl ester hydroperoxide lability is a key feature of the oxidative susceptibility of small, dense LDL.

Abundant evidence has been provided to substantiate the elevated cardiovascular risk associated with small, dense, low density lipoprotein (LDL) particles. The diminished resistance of dense LDL to oxidative stress in both normolipidemic and dyslipidemic subjects is established; nonetheless, the molecular basis of this phenomenon remains indeterminate. We have defined the primary molecular targets of lipid hydroperoxide formation in light, intermediate, and dense subclasses of LDL after copper-mediated oxidation and have compared the relative stabilities of the hydroperoxide derivatives of phospholipids and cholesteryl esters (CEs) as a function of the time course of oxidation. LDL subclasses (LDL1 through LDL5) were isolated from normolipidemic plasma by isopycnic density gradient ultracentrifugation, and their content of polyunsaturated molecular species of phosphatidylcholine (PC) and CE and of lipophilic antioxidants was quantified by reverse-phase high-performance liquid chromatography. The molar ratio of the particle content of polyunsaturated CE and PC species containing linoleate or arachidonate relative to alpha-tocopherol or beta-carotene did not differ significantly between LDL subspecies. Nonetheless, dense LDL contained significantly less polyunsaturated CE species (400 mol per particle) compared with LDL1 through LDL4 (range, approximately 680 to 490 mol per particle). Although the formation of PC-derived hydroperoxides did not vary significantly between LDL subspecies as a function of the time course of copper-mediated oxidation, the abundance of the C18:2 and C20:4 CE hydroperoxides was uniquely deficient in dense LDL (23 and 0.6 mol per particle, respectively, in LDL5; 47 to 58 and 1.9 to 2.3 mol per particle, respectively, in other LDL subclasses) at propagation half-time. When expressed as a lability ratio (mol hydroperoxides formed relative to each 100 mol of substrate consumed) at half-time, the oxidative lability of CE hydroperoxides in dense LDL was significantly elevated (lability ratio <25:100) relative to that in lighter, larger LDL particle subclasses (lability ratio >40:100) throughout the oxidative time course. We conclude that the elevated lability of CE hydroperoxides in dense LDL underlies the diminished oxidative resistance of these particles. Moreover, this phenomenon appears to result not only from the significantly elevated PC to free cholesterol ratio (1.54:1) in dense LDL particles (1.15:1 to 1.25:1 for other LDL subclasses) but also from their unique structural features, including a distinct apoB100 conformation, which may facilitate covalent bond formation between oxidized CE and apoB100.

Antioxidants↗
PubMed1998

Thermal stability of apolipoprotein B100 in low-density lipoprotein is disrupted at early stages of oxidation while neutral lipid core organization is conserved.

The time course of the unfolding characteristics of the protein moiety and of the thermotropic behavior of the core-located apolar lipids of highly homogeneous low-density lipoprotein (LDL) subspecies (d 1.030-1.040 g/mL) have been evaluated during transition metal- and azo radical-induced oxidation using differential scanning calorimetry. Apolipoprotein B100 (apo-B100) structure was highly sensitive to oxidative modification; indeed, a significant loss of thermal stability was observed at initial stages irrespective of whether oxidation was mediated by site-specific binding of copper ions or by free radicals generated during decomposition of azo compounds. Subsequently, thermal protein integrity was destroyed, as a result of potentially irreversible protein unfolding, cross-linking reactions, and aggregation. Our results suggest that even minimal oxidative modification of apo-B100 has a major impact on the stability of this large monomeric protein. By contrast, the core lipids, which consist primarily of cholesteryl esters and triglycerides and play a determinant role in the thermal transition occurring near physiological temperature, preserved features of an ordered arrangement even during propagation of lipid peroxidation.

Amidines↗
PubMed1996

Crystallization and preliminary X-ray analysis of a low density lipoprotein from human plasma.

Single crystals of human plasma low density lipoprotein (LDL), the major transport vehicle for cholesterol in blood, have been produced with a view to analysis of the three-dimensional structure by x-ray crystallography. Crystals with dimensions of approximately 200 x 100 x 50 microm have been reproducibly obtained from highly homogeneous LDL particle subspecies, isolated in the density ranges d = 1.0271-1. 0297 g/ml and d = 1.0297-1.0327 g/ml. Electron microscopic imaging of ultrathin-sectioned preparations of the crystals confirmed the existence of a regular, quasihexagonal arrangement of spherical particles of approximately 18 nm in diameter, thereby resembling the dimensions characteristic of LDL after dehydration and fixation. X-ray diffraction with synchrotron radiation under cryogenic conditions revealed the presence of well resolved diffraction spots, to a resolution of about 29 A. The diffraction patterns are indexed in terms of a triclinic lattice with unit cell dimensions of a = 16. 1 nm, b = 39.0 nm, c = 43.9 nm; alpha = 96.2 degrees, beta = 92.1 degrees, gamma = 102 degrees, and with space group P1.

Crystallography, X-Ray↗
PubMed1995

Core lipid structure is a major determinant of the oxidative resistance of low density lipoprotein.

The influence of thermally induced changes in the lipid core structure on the oxidative resistance of discrete, homogeneous low density lipoprotein (LDL) subspecies (d, 1.0297-1.0327 and 1.0327-1.0358 g/ml) has been evaluated. The thermotropic transition of the LDL lipid core at temperatures between 15 degrees C and 37 degrees C, determined by differential scanning calorimetry, exerted significant effects on the kinetics of copper-mediated LDL oxidation expressed in terms of intrinsic antioxidant efficiency (lag time) and diene production rate. Thus, the temperature coefficients of oxidative resistance and maximum oxidation rate showed break points at the core transition temperature. Temperature-induced changes in copper binding were excluded as the molecular basis of such effects, as the saturation of LDL with copper was identical below and above the core transition. At temperatures below the transition, the elevation in lag time indicated a greater resistance to oxidation, reflecting a higher degree of antioxidant protection. This effect can be explained by higher motional constraints and local antioxidant concentrations, the latter resulting from the freezing out of antioxidants from crystalline domains of cholesteryl esters and triglycerides. Below the transition temperature, the conjugated diene production rate was decreased, a finding that correlated positively with the average size of the cooperative units of neutral lipids estimated from the calorimetric transition width. The reduced accessibility and structural hindrance in the cluster organization of the core lipids therefore inhibits peroxidation. Our findings provide evidence for a distinct effect of the dynamic state of the core lipids on the oxidative susceptibility of LDL and are therefore relevant to the atherogenicity of these cholesterol-rich particles.

Calorimetry, Differential Scanning↗
PubMed1994

Lipoprotein (a): implication in atherothrombosis.

Substantial experimental evidence now implicates lipoprotein (a) as an independent risk factor for premature cardiovascular disease. Both plasma Lp(a) levels and apo(a) phenotype are strong predictors of risk for ischaemic heart disease. The accumulation of apo(a) in vascular wall tissue and in atherosclerotic plaques and the potential inhibition of fibrinolysis by Lp(a) underlie the enhanced risk of premature cardiovascular disease associated with this cholesterol-rich particle. Recent studies of the capacity of purified Lp(a) isoforms to inhibit fibrinolysis in an in vitro system have revealed that small isoforms of Lp(a) (< or = 500 kDa) are efficient inhibitors of plasminogen activation and bind with high affinity to fibrin. Conversely, large isoforms exert little or no inhibitory effect in this system (> 500 kDa). These data suggest that the potential, high affinity interaction of Lp(a) particles containing small isoforms with fibrin introduces a new, third dimension to the atherothrombotic risk associated with these cholesterol-rich particles.

Apolipoproteins A↗
PubMed1991

Cytokine production by cholesterol-loaded human peripheral monocyte-macrophages: the effect on fibrinogen mRNA levels in a hepatoma cell-line (HepG2).

Conditioned medium from human monocyte-macrophages incubated under various conditions was tested for its ability to stimulate fibrinogen mRNA levels in the hepatoma cell line HepG2. Recombinant human interleukin-6 (IL-6) stimulated fibrinogen mRNA levels 4.4-fold over control levels; this response was blocked by an anti-IL-6 antibody. Conditioned medium from 3-day-cultured monocyte-macrophages produced a slight stimulation of fibrinogen synthesis in HepG2 cells which was enhanced when the monocyte-macrophages had been treated with lipopolysaccharide (LPS). This stimulation was blocked by the anti IL-6 antibody. The cytokines, interleukin-1 (IL-1) and tumour necrosis factor (TNF) were also detected in the conditioned medium from the 3-day-cultured monocyte-macrophages. Monocyte-macrophages were cultured for 17 days and then incubated with acetylated low density lipoprotein (AcLDL) for 48 h. Such cells were 'foamy' in appearance and showed a 4-fold increase in apoE mRNA and a 10 to 50-fold increase in apoE secretion. This increase in apoE production was suppressed by almost a third when cells were coincubated with AcLDL and LPS. Conditioned medium from these 17-day-cultured AcLDL-treated human monocyte-macrophages did not stimulate fibrinogen mRNA synthesis in HepG2 cells, nor did the conditioned medium contain detectable levels of cytokines. These results suggest that cytokine production from foam cells in the atherosclerotic lesion is unlikely to be a major contributing factor in determining the elevated fibrinogen levels seen in the plasma of patients with IHD.

Antibodies↗
PubMed1991

Native low-density lipoproteins stimulate leukotriene B4 production by human monocyte-derived macrophages.

We have evaluated the effect of native low-density lipoproteins (LDL) on the production of leukotriene B4 (LTB4), a potent inflammatory and chemotactic factor, by human monocyte-derived macrophages. The capacity of LDL (d, 1.024-1.050 g/ml) to increase LTB4 secretion was dose-dependent with an optimal response at 100 micrograms LDL protein/ml, representing an approx. 7.5-fold stimulation over basal levels at 10 days of culture; the half-maximal response occurred at 20 micrograms/ml. The effect of LDL on LTB4 production was rapid (within 15 min) and was maintained for at least 21 h. The generation of LTB4 in response to LDL was partially inhibited (approx. 70% inhibition) by EDTA (5 mM) and by a monoclonal antibody (IgG-C7; 160 micrograms/ml) directed against the binding site of the cellular LDL receptor. In addition, the effects of native LDL and acetylated LDL were additive. These findings suggest that the specific interaction of LDL with its high affinity receptor represents a major component in the stimulation of the production of LTB4 by human monocyte-derived macrophages.

Antibodies, Monoclonal↗
PubMed1991

Discrete subspecies of human low density lipoproteins are heterogeneous in their interaction with the cellular LDL receptor.

The low density lipoproteins (LDL) of human plasma consist of a series of discrete particle subspecies of distinct physicochemical, immunological, and hydrodynamic properties. Such structural differences are intimately linked to the metabolic heterogeneity of circulating LDL in vivo. The current studies were designed to evaluate and compare the interaction of discrete LDL subspecies from normolipidemic subjects with the LDL receptor. Plasma LDL of d 1.019-1.063 g/ml from healthy males were fractionated into 15 subspecies of defined physicochemical characteristics by isopycnic density gradient ultracentrifugation as described earlier (Chapman et al., J. Lipid Res. 1988. 29: 442-458). The major LDL subspecies, LDL-5 to LDL-10, exhibited an overall range in density from 1.0244 to 1.0435 g/ml; individual subspecies increased in density by increments of 0.027 (LDL-5), 0.026 (LDL-6), 0.030 (LDL-7), 0.031 (LDL-8), 0.035 (LDL-9), and 0.042 g/ml (LDL-10), respectively. Taken together, these subspecies accounted for approximately 70% of the total mass of LDL of d 1.019-1.063 g/ml; their cholesterol: protein ratios decreased from 1.70 to 1.12 and particle size from 275 to 260 A with increase in density. ApoB-100 was the unique protein component in subspecies 5-8, with trace amounts (less than 0.2% of apoLDL) of both apoA-I and apoE in subspecies 9 and 10. The interaction of individual LDL subspecies with the LDL receptor on cultured human U-937 monocyte-like cells was compared by determining receptor binding affinities at 4 degrees C. Scatchard analysis of specific binding curves demonstrated a single class of binding site for each subspecies. The lowest dissociation constants were displayed by LDL subspecies 6 (Kd 5.71 nM), 7 (Kd 5.24 nM) and 8 (Kd 4.67 nM), while subspecies 5, 9, and 10 displayed significantly higher Kd values (8.35, 7.20, and 6.87 nM, respectively). Competitive displacement studies at 4 degrees C, in which unlabeled subspecies from the same gradient series competed for binding with 125I-labeled LDL subspecies, confirmed the relative binding affinities of these subspecies. As the hydrophobic lipid core of LDL undergoes a thermotropic transition at approximately 37 degrees C, which may in turn influence the surface structure of the particle, internalization and degradation studies were performed at 37 degrees C. No effect of temperature was detectable; again, LDL subspecies at each extreme of the density distribution (LDL-5 and LDL-10) displayed significantly lower binding affinities for the LDL receptor than that from the peak region (LDL-7).(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1990

Apolipoprotein E expression by human-monocyte-derived macrophages. Modulation by opsonised zymosan and cholesterol.

The effects of opsonised zymosan and of acetylated low-density lipoprotein (AcLDL) on the synthesis and secretion of apolipoprotein E (apoE), and of apoE mRNA abundance, have been studied in human-monocyte-derived macrophages (MDM). Stimulation by opsonised zymosan led to a concentration-dependent increase in apoE secretion; non-opsonised zymosan was without effect. Incubation with AcLDL led to a concentration-dependent elevation in apoE synthesis which paralleled the increase in cellular cholesterol content. The opsonised-zymosan-induced stimulation of apoE production was additive to that resulting from cholesterol loading with AcLDL. Opsonised zymosan alone did not affect the cholesterol content of MDM. Cholesterol-loaded MDM remained responsive to opsonised zymosan stimulation, displaying a 3.5-fold elevation in apoE secretion as compared to their non-stimulated counterparts. Cell-associated apoE remained at trace levels under all conditions of cell treatment. Studies involving [35S]methionine incorporation showed de novo synthesis of apoE to be enhanced in both cholesterol-loaded and opsonised-zymosan-stimulated macrophages. Estimation of apoE mRNA in opsonised-zymosan-stimulated and control MDM by dot-blot analysis revealed similar message abundance; by contrast, elevation in cellular cholesterol content following incubation with modified LDL led to a significant increase in apoE mRNA levels. We conclude that the opsonised-zymosan-induced stimulation of apoE synthesis and secretion in human MDM may occur by a mechanism(s) independent of cellular cholesterol content.

Apolipoproteins E↗
PubMed1988

Platelet activating factor is a potent stimulant of the production of active oxygen species by human monocyte-derived macrophages.

Platelet activating factor (PAF; C16), 1-O-Hexadecyl-2-acetyl-sn-glycero-3-phosphorylcholine) stimulated the production of active oxygen species by human monocyte-derived macrophages in culture. An optimal response was observed at a concentration of 13 microM PAF with half-maximal stimulation at 5 microM. The generation of superoxide ion (O2-) and hydrogen peroxide (H2O2) in response to PAF was inhibited specifically by a PAF-antagonist (1-O-Hexadecyl-2-acetyl-sn-glycero-3-phospho (N,N,N,-trimethyl) hexanolamine; such generation varied with the degree of maturation of cultured monocytes into macrophages. Production of active oxygen species increased progressively to reach a maximal level between days 4 to 6 of culture and remained maximal to day 12, after which it decreased progressively. Phorbol 12-myristate-13-acetate (PMA) and opsonized zymosan also stimulated generation of O2- and H2O2. PAF was however distinguished by its potent capacity to stimulate O2- and H2O2 production even at late stages of macrophage maturation (18 days), at which time both PMA and zymosan lacked significant effect. These findings suggest that PAF is a factor of potential relevance to the inflammatory role of the macrophage in atherogenesis.

Cell Differentiation↗
PubMedDate not supplied

Expression of elastase activity by human monocyte-macrophages is modulated by cellular cholesterol content, inflammatory mediators, and phorbol myristate acetate.

We investigated the effects of a number of stimulatory agents on the production of both cell-associated and extracellular elastase-type enzymes on human monocyte-macrophages in vitro and of the modulation of such effects by modification of cellular cholesterol content. The stimulatory agents included phorbol myristate acetate (PMA) and the inflammatory mediators, lipopolysaccharide (LPS), opsonized zymosan (OZ), and platelet activating factor (PAF). Using the synthetic substrate, N-succinyl-trialanyl-paranitroanilide (SANA), we detected cell-associated elastase-like activity in monocyte-derived macrophages. Such activity increased markedly with cell maturation over the period from 5 to 15 days of adherence culture. While PAF (10 micrograms/ml) and LPS (10 micrograms/ml) were without effect on cell-associated elastase-like activity in macrophages, PMA (100 ng/ml) and OZ (1 mg/ml) markedly stimulated such activity in cells cultured for 15 days. Furthermore, a fivefold increase in the cell-associated elastase-like activity of macrophages occurred upon cholesterol loading of the cells with acetylated low density lipoprotein (AcLDL). By contrast, this activity was markedly diminished upon depletion of cellular cholesterol content after incubation with high density lipoprotein (HDL3). Latent elastinolytic activity in the culture medium was detected by use of a radioactive substrate, insoluble 3H-elastin, after initial tryptic treatment of the medium. Such latent elastase activity was secreted only by activated macrophages; the relative potency of stimulation was: PMA greater than LPS = PAF greater than OZ. Increase in cellular cholesterol content alone markedly enhanced the secretion of elastase (from undetectable levels to 28 ng of 3H-elastin degraded/hr/micrograms DNA). In all cases, both the cell-associated and secreted latent elastinolytic activities were due to metalloproteases, in view of their 90% inhibition by 2 mM EDTA. Cholesterol-loaded macrophages, which displayed an approximately 40-fold increase in total cholesterol content as compared to control cells, remained sensitive to the action of activators of OZ and PMA, while LPS and PAF exerted only weak effects. Our data indicate that cellular cholesterol content and inflammatory mediators are effective stimulants of the production and secretion of elastase-type enzymes by human monocyte-macrophages. Among these factors, cellular cholesterol content, OZ, PAF, and LPS may represent factors of relevance to the inflammatory role of the macrophage in atherogenesis and more specifically to the alteration of elastin structure in the extracellular matrix of the vessel wall.

Cells, Cultured↗