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T Mazzone

Publications and source records attributed to T Mazzone.

At least 19 recordsLinked to original sources
PubMed2001

Sterol efflux mediated by endogenous macrophage ApoE expression is independent of ABCA1.

Sterol efflux importantly contributes to preservation of cellular cholesterol homeostasis, and multiple pathways may be involved for mediating such efflux. Recently, an important role has been ascribed to ABCA1 in facilitating lipid efflux from cells, including macrophages, to extracellular lipid-free apolipoproteins. Macrophages are relatively unique among cells because they express apoprotein E (apoE) as a major protein product, and this endogenous expression of apoE increases sterol and phospholipid efflux from macrophages. The studies in this article were designed to test whether the sterol efflux mediated by the endogenous expression of apoE in macrophages was dependent on ABCA1 expression. These studies were facilitated by comparing apoE-expressing J774 cells (J774E(+)) with nonexpressing parental cells (J774E(-)). Sterol efflux was higher from J774E(+) cells compared with J774E(-) cells, but the increment in efflux between these cell types was not increased by induction of ABCA1 expression with cAMP. Induction of ABCA1 with cAMP, however, did increase sterol efflux to exogenously added apoA1 from both cell types. Inhibitors of ABCA1 activity significantly reduced (by 40% to 50%) sterol efflux from both J774E(+) and J774E(-) cells treated with cAMP and apoA1. This inhibitor did not, however, reduce the increment in sterol efflux due to the expression of endogenous apoE. The results of these studies indicate that the increment in sterol efflux mediated by the endogenous expression of apoE in macrophages does not depend on ABCA1 expression or activity.

ATP Binding Cassette Transporter 1↗
PubMed2001

Increased prevalence of significant coronary artery calcification in patients with diabetes.

OBJECTIVE: Coronary artery disease is the major cause of morbidity and mortality in patients with diabetes. Detection of coronary artery disease before the first myocardial infarction and before anginal symptoms will allow for strategies designed to reduce the cardiovascular event rate in this group of patients. Electron beam-computed tomography (EBCT) is a noninvasive technology for evaluating the extent of coronary artery atherosclerosis that relies on the detection of coronary artery calcium (CAC). We used EBCT to detect significant coronary artery atherosclerosis in diabetic patients without symptoms of heart disease. RESEARCH DESIGN AND METHODS: We used EBCT to evaluate calcium in the coronary arteries of 139 consecutive diabetic patients scanned over a 20-month period. The CAC scores in this group were compared with a randomly selected nondiabetic control group and a control group that was selected to match a number of established cardiovascular risk factors. RESULTS: Patients with diabetes had a significant increase in the prevalence of CAC scores > or =400 (25.9%) compared with the randomly selected (7.2%) and matched (14.4%) nondiabetic control groups. Scores in this range have been reported to be highly predictive for abnormal stress myocardial perfusion tomography and subsequent coronary events. CONCLUSIONS: Our results, therefore, indicate a substantial prevalence of significant coronary artery disease in an asymptomatic diabetic patient population compared with nondiabetic control subjects. They also suggest that EBCT may be a useful approach for selecting a group of diabetic subjects who would benefit most from additional evaluation for subclinical coronary artery disease.

Calcinosis↗
PubMed2001

Interaction with proteoglycans enhances the sterol efflux produced by endogenous expression of macrophage apoE.

Endogenous expression of apolipoprotein (apo)E in macrophages facilitates cholesterol efflux in the presence and absence of extracellular sterol acceptors. A proteoglycan-associated pool of apoE has also been described. The relationship between a proteoglycan-associated pool of apoE and enhanced cholesterol efflux was investigated in these studies. Inhibition of proteoglycan expression reduced cholesterol efflux from apoE-expressing cells ( J774E(+)) in the presence and absence of HDL, but did not do so from nonexpressing cells ( J774E(-)). The effect of proteoglycan depletion on sterol efflux from J774E(+) cells was confirmed by measuring differences in cell sterol mass, secreted sterol mass, and sterol efflux rates. Furthermore, apoE-containing particles secreted from proteoglycan-depleted J774E(+) cells were denser than those secreted from J774E(+) cells with intact proteoglycan expression. Also, in J774E(+) cells with intact proteoglycans, apoE particles isolated from the cell surface proteoglycan layer were denser than secreted particles. The apoE-lipid particles isolated from the cell surface proteoglycan layer had a lower lipid-to-apoE and cholesterol-to-apoE ratio compared with secreted particles. In distinction, proteoglycan depletion of J774E(-) cells did not reduce sterol efflux produced by the exogenous addition of apoE. These observations indicate that one mechanism by which endogenous expression of apoE facilitates effective cholesterol efflux from macrophages is related to its retention at the cell surface in a proteoglycan-associated pool. Further, our data suggest that apoE arrives at the cell surface in a relatively lipid-poor state, and that a proximate source of lipid available to the proteoglycan-bound apoE at the cell surface resides in the plasma membrane.

Animals↗
PubMed2000

Endogenously expressed apolipoprotein E has different effects on cell lipid metabolism as compared to exogenous apolipoprotein E carried on triglyceride-rich particles.

Apolipoprotein E (apoE) on model triglyceride-rich particles (TGRP) increases triglyceride (TG) utilization and cholesteryl ester (CE) hydrolysis, independent of its effect on enhancing particle uptake. We questioned whether, under physiological concentrations, endogenously expressed apoE has similar effects on cellular lipid metabolism as compared to exogenous apoE. J774 macrophages, which do not express apoE, were engineered to express endogenous apoE by transfection of human apoE3 cDNA expression constructs (E(+)) or control vectors (E(-)) into the cells. To compare the effects of exogenous apoE and endogenous apoE on TGRP uptake, cells were incubated with or without apoE associated with (3)H-cholesteryl ether-labeled TGRP. Exogenous apoE enhanced TGRP uptake in both E(-) and E(+) cells. E(-) cells displayed significantly higher TGRP uptake than E(+) cells. Sodium chlorate, which inhibits cell proteoglycan synthesis, markedly diminished differences in TGRP uptake between E(-) and E(+) cells, suggesting that endogenous apoE-proteoglycan interaction contributes to differences in uptake between the two cell types. Particle uptake by the LDL receptor, by the LDL receptor related protein, or by scavenger receptors were similar between E(-) and E(+) cells indicating that endogenous apoE expression does not have a general effect on endocytic pathways. Exogenous apoE carried on TGRP stimulated TG utilization and CE hydrolysis in both cell types. However, TG utilization and CE hydrolysis were not affected by endogenous apoE expression. In conclusion, macrophage expression of apoE has very different effects on TGRP metabolism than exogenously supplied apoE. The fluorescence microscopy results in this study showing that exogenous apoE and endogenous apoE were confined in separate cellular compartments support the hypothesis that these differences resulted from distinct intracellular trafficking pathways followed by exogenous apoE bound to TGRP as compared to endogenous cell-expressed apoE.

Apolipoproteins E↗
PubMed2000

Sterols and inhibitors of sterol transport modulate the degradation and secretion of macrophage ApoE: requirement for the C-terminal domain.

Macrophage-derived apoE, produced in the vessel wall, may have important effects during atherogenesis. The production of apoE by macrophages can be regulated at a transcriptional level by cellular differentiation state, cytokines and sterol loading. In addition, there are post-transcriptional and post-translational loci for regulation. We have recently identified an intermediate density cell membrane fraction in which the degradation of apoE can be modulated by sterols. Suppressing degradation of apoE in this fraction by pre-incubating cells in sterols led to enhanced apoE secretion. In this report we demonstrate that the suppressive effect of sterols on the degradation of newly synthesized apoE in this fraction depends on the presence on its C-terminal domain, by studying a macrophage cell line transfected to express a mutant form of apoE in which amino acids beyond amino acid 202 were deleted. In addition, two modulators of cellular sterol transport, progesterone and U1866A, inhibited the degradation of full-length apoE. In contrast, incubation of cells in the acyl-CoA:cholesterol acyltransferase inhibitor S58035 did not influence apoE degradation. As would be predicted based on the results of degradation assays, U1866A, but not S58035, increased the secretion of apoE from a cell line transfected to constitutively express full-length apoE cDNA. The effect of U1866A on apoE degradation, like the effect of sterol, required the presence of the apoE C-terminal domain. Our results indicate that alteration of intracellular sterol homeostasis by pre-incubation in sterols or by drugs that modify the subcellular transport of sterol, modulates the susceptibility of apoE to degradation and that this modulation requires the presence of C-terminal lipid binding domains.

Apolipoproteins E↗
PubMed2000

Transport and processing of endogenously synthesized ApoE on the macrophage cell surface.

We have previously established the presence of a pool of apoE sequestered on the macrophage cell surface by demonstrating its displacement from a cell monolayer at 4 degrees C. In this series of experiments, we use a cell surface biotinylation protocol to directly quantitate apoE on the macrophage cell surface and evaluate its transport to and from this cell surface pool. In human monocyte-derived macrophages labeled to equilibrium and in a mouse macrophage cell line transfected to constitutively express human apoE3, approximately 8% of total cellular apoE was present on the surface, but only a portion of this surface pool served as a direct precursor to secreted apoE. The half-life of apoE on the macrophage cell surface was calculated to be approximately 12 min. On SDS-polyacrylamide gel electrophoresis, the apoE isolated from the surface fraction of cells labeled to equilibrium migrated in an isoform pattern distinct from that observed from the intracellular fraction, with the surface fraction migrating predominantly in a higher molecular weight isoform. Pulse labeling experiments demonstrated that newly synthesized apoE reached the cell surface by 10 min but was predominantly in a low molecular weight isoform. There was also a lag between appearance of apoE on the cell surface and its appearance in the medium. Biotinylated apoE, which accumulated in the medium, even from pulse labeled cells, was predominantly in the high molecular weight isoform. Additional experiments demonstrated that low molecular weight apoE present on the cell surface was modified to higher molecular weight apoE by the addition of sialic acid residues prior to secretion and that this conversion was inhibited by brefeldin A. These results demonstrate an unexpected complexity in the transport and cellular processing of macrophage cell surface apoE. Factors that modulate the size and turnover of the cell surface pool of apoE in the macrophage remain to be identified and investigated.

Animals↗
PubMed2000

The heparin-binding proteins apolipoprotein E and lipoprotein lipase enhance cellular proteoglycan production.

Apolipoprotein E (apoE) and lipoprotein lipase (LPL), key proteins in the regulation of lipoprotein metabolism, bind with high affinity to heparin and cell-surface heparan sulfate proteoglycan (HSPG). In the present study, we tested whether the expression of apoE or LPL would modulate proteoglycan (PG) metabolism in cells. Two apoE-expressing cells, macrophages and fibroblasts, and LPL-expressing Chinese hamster ovary (CHO) cells were used to study the effect of apoE and LPL on PG production. Cellular PGs were metabolically labeled with (35)[S]sulfate for 20 hours, and medium, pericellular PGs, and intracellular PGs were assessed. In all transfected cells, PG levels in the 3 pools increased 1.6- to 3-fold when compared with control cells. Initial PG production was assessed from the time of addition of radiolabeled sulfate; at 1 hour, there was no difference in PG synthesis by apoE-expressing cells when compared with control cells. After 1 hour, apoE-expressing cells had significantly greater production of PGs. Total production assessed with [(3)H]glucosamine was also increased. This was due to an increase in the length of the glycosaminoglycan chains. To assess whether the increase in PGs was due to a decrease in PG degradation, a pulse-chase experiment was performed. Loss of sulfate-labeled pericellular PGs was similar in apoE and control cells, but more labeled PGs appeared in the medium of the apoE-expressing cells. Addition of exogenous apoE and anti-human apoE antibody to both non-apoE-expressing and apoE-expressing cells did not alter PG production. Moreover, LPL addition did not alter cell-surface PG metabolism. These results show that enhanced gene expression of apoE and LPL increases cellular PG production. We postulate that such changes in vascular PGs can affect the atherogenic potential of arteries.

Animals↗
PubMed1999

High vitamin A intake in early pregnancy and major malformations: a multicenter prospective controlled study.

The European Network of the Teratology Information Services (ENTIS) collected and evaluated data on 423 pregnancies exposed during the first 9 weeks of gestation to a "high" dose of vitamin A (10,000 IU per day or more). Data were collected prospectively; 394 women (93.1%) were followed by telephone interview up to the first few weeks after the expected date of delivery, using standardized procedures. The presence of major structural malformations, excluding chromosomal and genetic diseases, was evaluated in 311 infants exposed to a median daily dose of vitamin A of 50,000 IU per day (range, 10,000-300,000 IU per day; interquartile range, 25,000-60,000 IU per day). Three infants with a major malformation were reported: pulmonary stenosis, stenotic anus with fistula, and bilateral inguinal hernia. No congenital malformations were reported among 120 infants exposed to more than 50,000 IU per day of vitamin A. When the birth prevalence rate of major malformations in the study group was compared with two internal control groups of infants exposed to: 1) "high" vitamin A exposure later in pregnancy, and 2) nonteratogenic agent exposures, the rate ratio was, respectively, 0.28 (CI 95% interval, 0.06, 1.23) and 0.50 (CI 95% interval, 0.14, 1.76). The studied sample did not provide evidence for an increased risk of major malformations, associated with "high" vitamin A intake during the organogenetic period, higher than 2.76 above the control reference risk of 1.91% (power 80%, alpha 0.10).

Abnormalities, Drug-Induced↗
PubMed1999

Plasma lipids and stroke.

High plasma levels of lipids are an important modifiable risk factor for coronary heart disease, but are not established as a risk factor for stroke. Pathophysiologic evidence that links lipids to major systemic artery disease, and the results of clinical trials of coronary heart disease prevention in relation to lipid-lowering suggest that lipids may play an important role in the causation of stroke. We discuss the controversy concerning plasma lipids as a risk factor for stroke. A clinical trial targeted at lowering levels of lipids with the aim of primary stroke prevention would be a timely and important contribution. Armed with this information, we could further clarify the plasma lipid-stroke controversy and move into the 21st century with a better understanding of stroke prevention.

Humans↗
PubMed1999

LDL receptor binds newly synthesized apoE in macrophages. A precursor pool for apoe secretion.

There appear to be multiple post-translational sites for regulation of macrophage apolipoprotein (apo)E secretion, including the presence of a distinct cell surface pool of apoE. Cell surface proteoglycans have been shown to be involved in forming this pool. The current studies were designed to investigate the role of an additional cell surface site, i.e., the low density lipoprotein (LDL) receptor. Antiserum to the LDL receptor displaced apoE from the macrophage cell surface and into the medium during a 4 degrees C incubation from apoE-expressing J774 cells, from proteoglycan-depleted apoE-expressing J774 cells, and from human monocyte-derived macrophages. Similar results were obtained when purified monoclonal antibody to the LDL receptor was added to human monocyte-derived macrophages. J774 cells transfected to express an LDL receptor binding-defective mutant of apoE did not show a similar response to addition of LDL receptor antibody. Studies were conducted in which cells were pulse labeled for 30 min, followed by various periods of chase at 4 degrees C or 37 degrees C in the presence or absence of LDL receptor antibody. The results of these studies indicated that nascent macrophage-derived apoE binds to the LDL receptor, and that this apoE served as a precursor pool for apoE released into the medium. These studies establish a role for the LDL receptor in forming the cell surface pool of apoE and, along with data regarding the importance of proteoglycans, indicate that cell surface binding sites for nascent macrophage-derived apoE are heterogeneous. The heterogeneity of such sites could have implication for the size and turnover of this cell surface pool.

Animals↗
PubMed1999

Apolipoprotein E-dependent cholesterol efflux from macrophages: kinetic study and divergent mechanisms for endogenous versus exogenous apolipoprotein E.

In these studies, we have utilized a J774 macrophage model in order to compare phospholipid and cholesterol efflux kinetics in macrophage cells that do not express endogenous apoE to cells transfected to express physiologic levels of human apoE. This model was also used to compare the effect of exogenously added versus endogenously expressed apoE on cholesterol efflux kinetics from macrophages. ApoE expression increased free cholesterol and phospholipid efflux into the medium, but did not change the free cholesterol/phospholipid molar ratio of secreted lipids. Kinetic examination showed that free cholesterol and phospholipid appeared simultaneously in the medium, and that cholesterol loading widened the difference in the rate of cholesterol efflux between apoE-expressing and non-expressing macrophages. Addition of exogenous lipid-free apoE added to non-expressing cells, at a >2-fold higher medium concentration than that produced by endogenous expression, produced less cholesterol efflux than that observed from apoE-expressing cells. The addition of phosphatidylcholine liposomes substantially increased cholesterol efflux from apoE-expressing and non-expressing J774 cells. Addition of these liposomes eliminated the enhanced cholesterol efflux produced by addition of exogenous apoE. On the other hand, even in the presence of phosphatidylcholine liposomes, cholesterol efflux rates remained significantly higher from apoE-expressing macrophages than non-expressing cells. Similar results were obtained when efflux was studied in the presence of cyclodextrin. These results suggest that endogenous expression of apoE by macrophages alters cell cholesterol balance via mechanisms distinct from those utilized by the extracellular addition of apoE, and may involve intracellular or pericellular mechanisms.

Animals↗
PubMed1998

The safety of omeprazole during pregnancy: a multicenter prospective controlled study.

OBJECTIVES: Our purpose was to determine whether omeprazole use during pregnancy is associated with an increased risk of malformations, spontaneous abortions, decreased birth weight, or perinatal complications. STUDY DESIGN: In a multicenter, prospective controlled study, pregnant women exposed to omeprazole during gestation were matched with controls exposed to nonteratogens and with disease-paired controls who used histamine blockers for similar indications. The primary end point was the incidence of major malformations. RESULTS: One hundred thirteen pregnant women were exposed to omeprazole during pregnancy. Rates of major malformations in the omeprazole group (4%) did not differ from controls exposed to nonteratogens (2%) (P = .68, relative risk = 1.94, 95% confidence interval 0.36 to 10.36) and disease-paired controls (2.8%). Birth weight, gestational age at delivery, preterm deliveries, and neonatal complications were comparable among the three groups. CONCLUSIONS: No association was found between exposure to omeprazole during the period of organogenesis and increased risk for major malformations. Exposure throughout pregnancy is not associated with increased risk of spontaneous abortions, decreased birth weight, or perinatal complications.

Abnormalities, Drug-Induced↗
PubMed1998

A prospective controlled multicentre study of clarithromycin in pregnancy.

Clarithromycin is a relatively new macrolide antibiotic with an action spectrum similar to that of erythromycin. Its main indications for use are for upper and lower respiratory and skin and soft tissue infections. Little is known about its safety in pregnancy, although animal reproductive studies found an increased rate of cardiovascular anomalies, cleft palate, and embryonic loss. Human data, limited to case reports and one small uncontrolled study, cannot allow evidence based counseling of pregnant women who were exposed to the drug before finding out they were pregnant. Pregnant women who had been counseled on the use of clarithromycin by five centers, were matched for age, smoking, and alcohol use with a control group of pregnant women who were exposed to nonteratogenic antibiotics. A total of 157 women were followed up. Of these, 122 were exposed to the drug in the first trimester. There were no significant differences found between the two groups in the rates of major and minor malformations; 2.3 versus 1.4% for major (p = 0.86) and 5.4 versus 4.9% for minor (p = 0.96). Spontaneous abortion rates in the exposed group was significantly different, higher (14%) than in the control group (7%) (p = 0.04). This first prospective controlled study of exposure to clarithromycin in pregnancy suggests that this agent does not increase the rate of major malformations above the baseline risk of 1-3%. The higher rate of reported spontaneous abortions, although still within the expected baseline rate, may warrant further study.

Anti-Bacterial Agents↗
PubMed1998

Endogenous apoE expression modulates HDL3 binding to macrophages.

We have previously shown that expression of a human apoE cDNA in J774 macrophages enhances cholesterol efflux to HDL3. We have also shown that endogenous apoE expression produces a cell surface pool of apoE associated with proteoglycans. In this series of experiments, we first demonstrate the presence of a cell surface proteoglycan-associated apoE pool in human monocyte-derived macrophages. We then examine the hypothesis that endogenous expression of apoE modulates HDL3 binding to macrophages, thereby, accounting for enhanced cholesterol efflux to HDL3, specifically examining a role for the cell surface pool. Enhanced binding of apoE-free human HDL3 to apoE-expressing macrophages, compared to non-expressing macrophages, was observed at 37 degrees C and 4 degrees C. The enhanced binding was not due to apoE secreted into the medium, as determined by experiments utilizing conditioned medium from apoE-secreting cells. Removal of the cell surface pool of apoE, however, substantially reduced the incremental HDL3 binding produced by apoE expression. Cellular cholesterol mass measurements demonstrated that experimental conditions that reduced HDL3 binding to apoE-expressing macrophages, did not substantially reduce cholesterol efflux to HDL3. In summary, our results document a clear role for cell surface pool of apoE in modulating HDL3 interaction with macrophages. The enhanced binding, however, does not appear to be a major mechanism contributing to the increased cholesterol efflux to HDL3, which results from endogenous macrophage expression of apoE.

Apolipoproteins E↗
PubMed1997

Degradation of macrophage ApoE in a nonlysosomal compartment. Regulation by sterols.

Macrophage-derived apoE has been shown to play an important role in the susceptibility of the vessel wall to atherosclerosis. Previous studies have shown that macrophage sterol content modulates apoE synthesis and secretion, associated with a large transcriptional response of the apoE gene. The current studies were undertaken to evaluate the existence of additional post-transcriptional regulatory loci for the effect of sterols on apoE synthesis and secretion. Using a macrophage cell line transfected to constitutively express an apoE cDNA to facilitate detection of a post-transcriptional regulatory locus, we demonstrated that preincubations in 25-hydroxycholesterol and cholesterol lead to increased apoE secretion in pulse/chase experiments. Examination of cell lysates in these experiments showed that apoE not secreted by control cells was degraded and not detectable, suggesting that the preincubation in sterols increased secretion by decreasing degradation of newly synthesized apoE. The measurement of total protein and apoE degradation in cell fractions revealed an intermediate density fraction that degraded significant amounts of newly synthesized total protein and newly synthesized apoE. In this fraction, degradation of total protein and apoE was unaffected by chloroquine but was substantially reduced by N-acetyl-Leu-Leu-norleucinal plus N-acetyl-Leu-Leu-methioninal or by lactacystin, suggesting the involvement of proteasomes. Preincubation in sterol/oxysterol or acetylated low density lipoprotein did not modify total protein degradation by this fraction but inhibited apoE degradation. Similar results were obtained using intermediate density fractions isolated from human monocyte-derived macrophages. The results of our studies indicate that newly synthesized apoE in the macrophage can be degraded in an intermediate density nonlysosomal cellular compartment, which is sensitive to proteasomal inhibitors. Alteration of cellular lipid homeostasis by preincubation in sterol/oxysterol or acetylated low density lipoprotein inhibits apoE, but not total protein, degradation in this fraction. Inhibition of the degradation of apoE in this fraction likely contributes to the increased apoE secretion observed in sterol-enriched cells.

Apolipoproteins E↗