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C J Fielding

Publications and source records attributed to C J Fielding.

At least 91 records · Page 5Linked to original sources
PubMed1982

Regulation of sterol transport in human microvascular endothelial cells.

In cultured human dermal microvessel endothelial cells, the rate of efflux (about twofold greater than for fibroblasts under equivalent conditions) was coupled to an equivalent high rate of sterol net transport from the cells to the medium. This net transport was linked with esterification via lecithin:cholesterol acyltransferase. Since the use of free sterol by plasma transferase is constant, such increased net transport indicates that endothelial cells are highly efficient, in competition with plasma lipoproteins, in supplying free sterol for esterification. These results indicate the marked ability of endothelial cells to regulate and maintain their sterol balance in the face of high sterol levels to which these cells are uniquely exposed in human plasma.

Apolipoproteins↗
PubMed1982

Characterization and quantitation of apolipoproteins A-I and E of normal and cholesterol-fed guinea pigs.

We have characterized and quantified the two major plasma apoproteins of high density lipoproteins (HDL), apolipoproteins A-I (apoA-I) and E (apoE), of guinea pigs fed standard chow (normal) or chow supplemented with 1% cholesterol (cholesterol-fed). ApoA-I isolated from plasma HDL of the normal guinea pig exists in six polymorphic forms (pI 5.75-5.40). A similar isoform pattern of this apoprotein was present in nascent HDL isolated from perfused livers of normal and cholesterol-fed animals. This apoprotein contains cysteine and isoleucine and is slightly different in overall amino acid composition from apoA-I of human and rat, but activates lecithin:cholesterol acyltransferase from human plasma with an activation curve almost identical to that obtained with human apoA-I. ApoE present in nascent VLDL and HDL from perfused liver of normal animals contains three isoforms (pI 5.42-5.34). Following cholesterol feeding, the numbers of apoE isoforms from perfused livers were increased from three to five or more by shifting the major component (pI 5.42) to more acidic isoforms (pI 5.28-5.17). This shifting was mostly reversible when apoE was treated with neuraminidase, suggesting that cholesterol feeding leads to a modification of apoE by increasing its content of sialic acid. Similar changes of apoE isoforms were also observed in plasma lipoproteins as early as 10 days after cholesterol feeding. The amino acid compositions of four apoE isoform fractions isolated from plasma HDL of cholesterol-fed guinea pigs were similar to that of parent apoE. The plasma concentrations of apoA-I and apoE, measured by electroimmunoassay, were 6.2 +/- 2.0 and 2.2 +/- 0.5 mg/dl, respectively, in guinea pigs fed standard chow. In animals that had been fed 1% cholesterol, plasma levels of apoA-I slightly increased in 1 week and showed a twofold increase in 8-10 weeks. Plasma levels of apoE, on the other hand, sharply increased by 10-fold in 1 week and up to 22-fold in 8-10 weeks on the cholesterol diet.-Guo, L. S. S., R. L. Hamilton, J. P. Kane, C. J. Fielding, and G. C. Chen. Characterization and quantitation of apolipoproteins A-I and E of normal and cholesterol-fed guinea pigs.

Amino Acids↗
PubMed1981

Regulation of human plasma lecithin:cholesterol acyltransferase activity by lipoprotein acceptor cholesteryl ester content.

Very low density lipoproteins and low density lipoproteins attain maximal cholesteryl ester contents during the incubation of human plasma and, under these conditions, both lecithin:cholesterol acyltransferase and transfer proteins are inhibited. These lipoproteins provide the major part of free cholesterol for the lecithin:cholesterol acyltransferase reaction, and are the major acceptors of cholesteryl ester generated by lecithin:cholesterol acyltransferase and transported to the lipoprotein acceptors by the transfer protein. The results obtained indicate that the concentration of acceptor limits esterification and transfer in plasma, and that in vivo these acceptors contain close to their maximal cholesteryl ester content. Human plasma end product acceptor lipoproteins have a composition similar to that of the ester-rich large low density lipoprotein characteristic of primate models of experimental atherosclerosis.

Cholesterol Esters↗
PubMed1981

Monoglyceride hydrolase activities of rat plasma and platelets. Their properties and roles in the activity of lipoprotein lipase.

Rat plasma contains monoglyceride hydrolase activities against both 1(3)- and 2-monoglycerides. These activities are present as lipoprotein complexes recovered by either density flotation or by agarose gel chromatography with plasma high density lipoprotein. However neither activity is complexed with the major apoproteins (apo-A-I, apo-E) of this lipoprotein class. 2-Monoglyceride hydrolase (but not 1(3)-monoglyceride hydrolase) activity associates with the triglyceride-rich lipoprotein class. The two activities are also noncompetitive with respect to substrate, and differ in pH- and cofactor-dependence and sensitivity to inhibition by diethyl p-nitrophenyl phosphate. Rat platelets also contain both 1(3)- and 2-monoglyceride hydrolase activities. These differ in reactivity with antiesterase and after solubilization and electrophoretic migration with each other and with the corresponding plasma activities. Studies with the isolated perfused rat heart suggest that a major role in the catabolism of 2-monoglyceride generated from lipoprotein lipase activity at the coronary bed is played by the plasma 2-monoglyceride hydrolase activity.

Animals↗
PubMed1981

Evidence for a lipoprotein carrier in human plasma catalyzing sterol efflux from cultured fibroblasts and its relationship to lecithin:cholesterol acyltransferase.

Immunoaffinity chromatography has been used to study the determinants of sterol efflux and net transport from cultured fibroblasts to human plasma medium. Sterol efflux was highly (approximately 80%) dependent upon a minor lipoprotein fraction containing apolipoprotein A-I unassociated with other apolipoproteins. The remaining activity was associated with the lipoprotein-free fraction of plasma and could be replaced by apoprotein-free albumin. Efflux was independent of lecithin:cholesterol acyltransferase (EC 2.3.1.43) activity. Net transport (i.e., the excess of efflux over influx) was completely inhibited by inhibition of lecithin:cholesterol acyltransferase or its removal by affinity chromatography on immobilized antibodies to apolipoprotein A-I or D (components of the transfer complex in human plasma). In uninhibited plasma, efflux and net transport rates had similar kinetics, suggesting that these were linked functions and that net transport was initiated by a carrier-dependent efflux step that, in the absence of lecithin:cholesterol acyltransferase activity, was associated with an equivalent influx of free sterol to the cells and that, in the presence of lecithin:cholesterol acyltransferase, was associated with esterification and transfer protein activity. The cholesterol carrier lipoprotein function (approximately 5% of plasma apolipoprotein A-I) appears to be the first step of lecithin:cholesterol acyltransferase-linked sterol transport from cells.

Apolipoproteins↗
PubMed1981

The endothelium, triglyceride-rich lipoproteins, and atherosclerosis: insights from cell biology and lipid metabolism.

Endothelial cells in culture retain many of the functional properties of the endothelium in vivo. At high cell density, they become contact-inhibited. Endothelial cells, like vascular smooth muscle cells and fibroblasts, express binding sites for low density lipoprotein when depleted of sterol. In contact-inhibited endothelial cells (but not actively growing cells), a block to internalization is evident, so that the cells bind but do not interiorize low density lipoprotein. Lipoprotein sterol does not enter the cell or regulate endogenous sterol synthesis. On the other hand, both contact-inhibited and actively growing endothelial cells express a separate receptor for triglyceride-rich lipoproteins. Cholesterol in these lipoproteins, unlike that in LDL, effectively regulates sterol synthesis in contact-inhibited endothelial cells and also mediates the accumulation of cholesterol in these cells. These findings are related to current concepts of atherogenesis. Receptors for triglyceride-rich lipoproteins may promote cellular sterol accumulation, release of contact inhibition in the surrounding endothelial cells, and exposure of the underlying vascular smooth muscle cells to plasma concentrations of both triglyceride-rich lipoproteins and LDL.

Arteriosclerosis↗
PubMed1980

Characteristics of triacylglycerol and partial acylglycerol hydrolysis by human plasma lipoprotein lipase.

The rates of reaction of human lipoprotein lipase (EC 3.1.1.34) with triacylglycerol and partial acylglycerol substrates have been compared as a function of the concentration of lipase cofactor protein (apolipoprotein C-II). The data indicate that the dissociation constant for monoacylglycerol is approximately three orders of magnitude greater than for diacylglycerols, indicating that only when the concentrations of higher acylglycerols become vanishingly small will significant monoacylglycerol hydrolysis (from 1-monoacylglycerol generated by isomerization of the 2-substituted primary product) be mediated by the lipase. This is in spite of the fact that maximal reaction velocities with each of the potential substrates are similar. A 'lipolytic cycle' is proposed to explain binding and dissociation of substrates with cofactor-lipase complex during catabolism of triacylglycerols.

Apolipoproteins↗
PubMed1980

Interaction of lecithin:cholesterol acyltransferase and cholesteryl ester transfer protein in the transport of cholesteryl ester into sphingomyelin liposomes.

When isolated lecithin:cholesterol acyltransferase was incubated with cholesterol-lecithin liposomes in the presence of apolipoprotein A-1, cholesteryl ester accumulated until a maximal ester/lecithin weight ratio of 0.03 was reached. This was independent of the amount of enzyme present or the proportion of cholesterol relative to lecithin. The inhibition of transferase associated with accumulation of cholesteryl ester was relieved by additional lecithin-cholesterol liposomes but not by addition of sphingomyelin liposomes containing the same proportion of substrate unesterified cholesterol. These results indicate that it is the accumulation of cholesteryl ester product which directly inhibits transferase activity. When isolated cholesteryl ester transfer protein from human plasma was included in the reaction mixture, cholesteryl ester was transported to sphingomyelin-cholesterol liposomes, with associated release of transferase from product inhibition. Cholesteryl ester incorporated directly into the liposomes or synthesized from free cholesterol via the transferase reaction was equally transferred to sphingomyelin acceptor liposomes, indicating that the cholesteryl ester in these particles formed a single miscible pool for transfer.

Apolipoproteins↗
PubMed1980

A cholesteryl ester transfer complex in human plasma.

Immunoadsorption affinity chromatography has been used to define the structure of lipoproteins in human plasma containing lecithin:cholesterol acyltransferase (EC 2.3.1.43) (LCAT) and transfer protein (apo D). The whole of LCAT was absorbed by antibodies specific for apo D and for apo A-1, indicating that the enzyme is present in plasma exclusively as a complex with its cofactor (apo A-1) and product transfer protein (apo D). About 80% of apo D (but no LCAT) was removed by antibody to apo A-2, indicating the presence of most of apo D in the form of an enzyme-free complex will apo A-1 and apo A-2. After removal of LCAT with antibody to apo D, plasma was unreactive as a substrate with isolated LCAT, but substrate activity was generated by ultracentrifugal flotation with either intact or adsorbed plasma. The apparent stoichiometry of the complex with LCAT (LCAT:apo A-1:apo D) was 1.0:0.9:1.8; that of the complex containing apo A-1, apo A-2, and apo D was 3.9:2.2:1.0.

Apolipoprotein A-I↗
PubMed1979

Characteristics of chylomicron binding and lipid uptake by endothelial cells in culture.

Bovine vascular endothelial cells bind chylomicrons via a high affinity membrane receptor site. Subsequent to binding, the chylomicron apoprotein was neither internalized nor degraded by either sparse or confluent (contact-inhibited) cells. However, the adsorption of chylomicrons was associated with interiorization of chylomicron cholesteryl ester and triglyceride and the hydrolysis of these lipids to free cholesterol and unesterified fatty acids by a lysosome-dependent pathway. This pathway was active in both subconfluent and contact-inhibited cells. The chylomicron free cholesterol so produced inhibited endogeneous cholesterol synthesis measured in terms of the incorporation of [1-14C]-acetate into sterol. An excess of high density lipoprotein was 2- to 3-fold more effective in reducing both binding of chylomicrons and interiorization of chylomicron lipid than was low density lipoprotein. Chylomicron binding was not "down-regulated" by preincubation of the cells with low density lipoprotein or chylomicrons. The results are discussed in the context of cholesterol sources for contact-inhibited endothelial cells which do not interiorize low density lipoprotein cholesterol.

Animals↗
PubMed1979

Validation of a procedure for exogenous isotopic labeling of lipoprotein triglyceride with radioactive triolein.

A procedure has been developed for the exogenous isotopic labeling of triglyceride-rich lipoproteins (chylomicrons and very low density lipoproteins) using high specific activity radioactive triglyceride in the presence of aqueous dimethyl sulfoxide. The labeled product lipoproteins showed unchanged chemical and physical properties. When the particles had also been labeled biologically by incorporation of unesterified fatty acids into the triglycerides of lipoproteins secreted by liver or intestine, both endogenous and exogenous labels were removed at the same rates in the isolated perfused heart and liver or in intact or functionally hepatectomized rats. These experiments additonally indicated that the triglyceride fatty acid composition of chylomicrons and very low density lipoproteins was unchanged during triglyceride depletion in the peripheral tissues. Using such labeled lipoproteins it has been shown that uptake of remnant lipoprotein cholesteryl ester and triglyceride by the liver is simultaneous. The labeling procedure described should prove suitable for kinetic studies of the disposition of the various lipoprotein non-polar ('core') lipids.

Animals↗
PubMed1979

Effect of contact inhibition on the regulation of cholesterol metabolism in cultured vascular endothelial cells.

Cholesterol synthesis in actively growing bovine vascular endothelial cells is regulated by low density lipoprotein (LDL) at a step prior to mevalonate formation, in a manner comparable to that found in aortic smooth muscle cells. LDL uptake by these cells is associated with induction of cholesterol esterification, an increase in total cell cholesterol, and an inhibition of endogenous sterol synthesis. In contrast, cholesterol metabolism in confluent contact-inhibited endothelial cultures was not significantly affected by LDL even though the cells bind the lipoprotein at high affinity receptor sites. Lysosomal degradation and subsequent regulatory effects on cellular cholesterol metabolism, however, were observed in contact-inhibited endothelial cells incubated with cationized rather than native LDL. Cationized LDL enter the cells independently of the high affinity sites. Therefore, the primary regulation of cholesterol metabolism in these cells is neither through the appropriate intracellular enzymes nor through the high affinity surface receptors, but via an inhibition of LDL internalization. It is suggested that this inhibition is due to a strict contact-inhibited morphology which enables the endothelium of the larger arteries to function as a selective barrier to the high circulating levels of plasma LDL.

Animals↗
PubMed1978

Role of contact inhibition in the regulation of receptor-mediated uptake of low density lipoprotein in cultured vascular endothelial cells.

Bovine vascular endothelial cells during logarithmic growth bind, internalize, and degrade low density lipoprotein (LDL) via a receptor-mediated pathway. However, contact-inhibited (confluent) monolayers bind but do not internalize LDL. This is in contrast to aortic smooth muscle cells or endothelial cells that have lost the property of contact inhibition. These cells internalize and degrade LDL at both high and low cell densities. The LDL receptors of smooth muscle and sparse endothelial cells down-regulate in response to LDL. In contrast, normal endothelial cells at confluency show little response. When contact inhibition in endothelial monolayers was locally released by wounding, and LDL was present, only cells released from contact inhibition accumulated LDL cholesterol. In smooth muscle cells under the same conditions, the entire culture interiorized lipid. It thus appears that in endothelial cells, unlike smooth muscle cells, contact inhibition is the major factor regulating cellular uptake of LDL cholesteryl ester. Reversal of contact inhibition by wounding provides a mechanism by which the endothelium could be the primary initiator of the atherosclerotic plaque.

Aorta↗
PubMed1978

Isolation and characterization of a human serum cholesteryl ester transfer protein.

Human plasma has been shown to contain an apolipoprotein that mediates the transport of cholesteryl ester from high density lipoprotein (HDL) to very low density lipoprotein (VLDL) or low density lipoprotein (LDL). This activity, confined to the density greater than 1.063 g/ml interval, has been isolated from HDL and appears as a single migrating species by anionic or sodium dodecyl sulfate/polyacrylamide gel electrophoresis. It is unreactive to antibodies to the major HDL apolipoproteins. Antibodies prepared against this factor and immobilized on Sepharose remove the capacity of HDL and density greater than 1.21 g/ml fractions as well as whole plasma to transport cholesteryl ester from HDL. The system shows saturation kinetics with respect to plasma LDL and VLDL concentrations, and transport of cholesteryl ester was associated with reciprocal and equimolar back-transport of triglyceride from VLDL and LDL to the HDL fraction. The possible relationship of this apoprotein to apoprotein D is discussed.

Apolipoproteins↗
PubMed1978

Metabolism of cholesterol-rich chylomicroms. Mechanism of binding and uptake of cholesteryl esters by the vascular bed of the perfused rat heart.

The rate of uptake of cholesteryl ester from chylomicrons has been determined with the isolated perfused rat heart and both intact and functionally hepatectomized rats. Uptake was found to be proportional to the cholesteryl ester content of the particles. Transfer of cholesteryl ester to other lipoprotein classes of the plasma was negligible under these conditions, and loss of cholesteryl ester from the medium was associated with quantitative recovery in the vascular bed. The uptake mechanism was nonsaturable and independent of the lipoprotein lipase binding site. Compared with receptor-dependent uptake of low density lipoprotein cholesteryl ester by heart endothelium, the chylomicron pathway appears to provide a major proportion of cholesteryl ester cleared from the plasma. Uptake was initially heparin dependent, and cleared lipid was released by 10 microgram/ml of heparin; however, lipid taken up rapidly became heparin resistant and was then hydrolyzed slowly with production of unesterified fatty acid. These results are discussed in the context of the possible role of cholesterol-rich chylomicron remnant lipoproteins in atherogenesis.

Animals↗
PubMed1978

Metabolism of cholesterol-enriched chylomicrons. Catabolism of triglyceride by lipoprotein lipase of perfused heart and adipose tissues.

The chemical and biochemical properties of cholesterol-enriched and cholesterol-poor chylomicrons from rat lymph have been compared. The enriched particles, prepared from cholesterol-containing lipid dispersions, passed into the duodenum, had four to ten times the cholesteryl ester content of the control chylomicrons but had the same content of total "core" (cholesteryl ester + triglyceride) lipid. Both chylomicron species had the same protein composition, the same phospholipid composition, and the same composition of triglyceride fatty acids. The rate of hydrolysis of chylomicron triglyceride for enriched and control particles was determined using both soluble and membrane-supported lipoprotein lipase (LPL) species from heart and adipose tissues. The lipase that was functional in the isolated perfused heart showed no significant difference in initial catabolic rate with cholesterol-enriched and control chylomicrons. The same result was obtained with this isolated LPL species in vitro. The lipase that was functional in isolated perfused epididymal adipose tissue showed a slightly lower catabolic rate with cholesterol-enriched particles (84% of that obtained with control chylomicrons). The same result was obtained with isolated adipose tissue LPL. It is concluded that cholesteryl ester content of chylomicrons under these conditions neither affects their protein composition nor has a major effect on their rate of reaction with lipoprotein lipase.

Adipose Tissue↗
PubMed1977

Lipoprotein lipase. Isolation and characterization of a second enzyme species from postheparin plasma.

A lipoprotein lipase species (mol wt 69 250) has been isolated from rat postheparin plasma, which differs from the low-molecular-weight species previously characterized in its amino acid composition and hexosamine content, and in its lower affinity for triglyceride-rich lipoprotein substrates. However, both enzymes are activated by the same coprotein (C-terminal glutamic acid, apo-C-2) from human very low density lipoprotein and have a similar specificity for lipid esters. Neither purified enzyme is activated by heparin. Both are inhibited by molar sodium chloride. Both enzyme species can be recovered from the same plasma samples. The possible relationship of these proteins to the different functional lipoprotein lipase activities of muscle and adipose tissues is discussed.

Amino Acids↗