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A B S T R A C T Rat livers were perfused for 6 h without added plasma proteins using washed erythrocytes and buffer in a recirculating system. An inhibitor to the enzyme lecithin-cholesterol acyltransferase (5,5'-dithionitrobenzoic acid) was added in some experiments to prevent modification of substrate-lipids contained in secreted lipoproteins. The inhibitor did not detectably alter hepatic ultrastructure or gas exchange, but it inhibited the secreted lecithin-cholesterol acyltransferase by more than 85%. Very low density lipoproteins in perfusate were unaltered but the high density lipoproteins obtained from livers perfused with the inhibitor appeared disk-shaped in negative stain by electron microscopy with a mean edge thickness of 46±5 A and a mean diameter of 190±25 A. The high density lipoproteins were composed predominantly of polar lipids and protein with only small amounts of cholesteryl esters and triglycerides. The major apoprotein of these discoidal fractions had the same electrophoretic mobility as the arginine-rich apoprotein, whereas plasma high density lipoproteins contained mainly the A-I apoprotein. In all these respects the discoidal perfusate high density lipoproteins closely resemble those found in human plasma which is deficient in lecithin-cholesterol acyltransferase. Perfusate high density lipoproteins obtained in the absence of the enzyme inhibitor more closely resembled plasma high density lipoproteins in Portions of this work have appeared in an abstract and in a review (1, 2).Dr. Hamilton is the recipient of a Research Career Development Award. Dr. Williams was the recipient of a Postdoctoral Fellowship from the National Heart and Lung Institute. Dr. Fielding was an Established Investigator of the American Heart Association.Received for publication 30 January 1976 and in revised form 12 April 1976. chemical composition (content of cholesteryl esters and apoproteins) and in electron microscopic appearance. Purified lecithin-cholesterol acyltransferase synthesized cholesteryl esters at a substantially faster rate from substrate lipids of perfusate high density lipoproteins than those from plasma. The discoidal high density lipoproteins were the best substrate for this reaction. Thin sections of plasma high density lipoproteins indicated a spherical particle whereas discoidal high density lipoproteins stained with the characteristic trilaminar image of membranes. These observations suggest that the liver secretes disk-shaped lipid bilayer particles which represent both the nascent form of high density lipoproteins and preferred substrate for lecithin-cholesterol acyltransferase. INTRODUCTIONThe plasma lipoproteins are usually separated into four major physically defined groups although it is recognized that heterogeneity exists within each. This heterogeneity is further complicated by movements of both lipids and apoproteins between particles of the different groups. One approach to obtain a clearer understanding of the physiological significance of the different plasma lipoproteins h...

A minor fraction of plasma high-density lipoprotein (pre beta-1 HDL) has been shown to promote cholesterol efflux from peripheral cell membranes [Castro, G. R., & Fielding, C. J. (1988) Biochemistry 27, 25-29]. When isolated native plasma is incubated at 37 degrees C, this fraction is specifically decreased. On the other hand, the level of plasma pre beta-1 HDL is fully protected in the presence of even very low levels of fibroblasts, vascular smooth muscle cells, or macrophages. Blood cells were completely inactive in maintaining plasma pre beta-1 HDL levels in the absence of peripheral cells, even at the relatively high levels present in whole blood. The loss of pre beta-1 observed in isolated plasma was dependent upon lecithin-cholesterol acyltransferase (LCAT) activity. These data suggest that reverse cholesterol transport catalyzed by pre beta-1 HDL, and subsequent LCAT-mediated cholesterol esterification, is directly dependent upon the interaction between this HDL species and competent peripheral 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.A number of recent'studies have identified a pathway in several cultured cell lines by which low density lipoprotein (LDL) is taken up into the cells via a specific receptor (1-3y. After internalization, the lipoprotein is catabolized in the lysosomes. The lipoprotein apoprotein is degraded to low molecular weight material and the cholesteryl ester content is hydrolyzed by lysosomal cholesterol esterase and re-esterified by the acylCoA:cholesterol acyl-transferase pathway (1). Most of the studies reported so far have been carried out with cultured fibroblasts and smooth muscle cells. These cells are exposed under physiological conditions to only low concentrations of plasma lipoproteins such as are present in the intercellular lymph (4 The vascular endothelium in vivo reveals a characteristic appearance as a single layer of highly flattened, contact-inhibited cells. The use of the peptide fibroblast growth factor (FGF) (5) has now permitted the long-term maintenance-of pure endothelial clones from a variety of sources, including the coronary bed and aortic arch of several~species (6). These cells retain the in vivo properties of endothelium and have been used in the present research to define the significance of the LDL receptor-mediated pathway in vascular endothelium.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. MATERIALS AND METHODSPreparation of LDL and Lipoprotein-Deficient Serum. Human and bovine LDL (1.019 < density < 1.063 g/cm3), human very low density lipoprotein (VLDL) (density < 1.006 g/cm3), human high density lipoprotein (HDL) (1.063 < density < 1.21 g/cm3), and the homologous lipoprotein-deficient sera (LPDS) (density > 1.21 g/cm3) were obtained from...

The individual effects of dietary cholesterol and fat saturation on plasma lipoprotein concentrations were determined in an ethnically diverse population of normolipidemic young men (52 Caucasian, 32 non-Caucasian). The experimental diets contained -200 or 600 mg/d of cholesterol, 36-38% of calories as fat, and high or low proportions of saturated and polyunsaturated fat (polyunsaturated/saturated fat ratio -0.8 vs 0.3). At the lower cholesterol intake, the high saturated fat diet had only a modest effect on LDL cholesterol in Caucasians (+ 6 mg/di`) and none in non-Caucasians. 600 mg cholesterol with high saturated fat led to a substantial mean increase in LDL cholesterol, which was significantly greater in Caucasian than in non-Caucasian subjects (+ 31 mg/dl vs 16 mg/dl, P < 0.005). 600 mg cholesterol with increased polyunsaturated fat gave a mean LDL increase of 16 mg/dl, lower than found when the same high cholesterol intake was coupled with increased saturated fat. Variation in cholesterol rather than the proportions of saturated and polyunsaturated fat had the most influence on LDL-cholesterol levels. Among non-Caucasians it was the only significant factor. (J. Clin. Invest. 1995. 95:611-618.)

IntroductionHuman (Hu) lecithin-cholesterol acyltransferase (LCAT) is a key enzyme in the plasma metabolism of cholesterol. To assess the effects of increased plasma levels of LCAT, four lines of transgenic mice were created expressing a Hu LCAT gene driven by either its natural or the mouse albumin enhancer promoter. Plasma LCAT activity increased from 1. The physiologic substrates for LCAT are nascent and mature HDL. Human (Hu) HDL is very heterogeneous both in particle size (5) and protein composition (6). HDL can be separated into two subpopulations on the basis of protein composition, one containing both apo AI and apo All (LpAI/AHI) and one containing apo AI but no apo All (LpAI). Both are heterogeneous in size (7). The size heterogeneity of Hu HDL distinguishes it from the monodisperse population of HDL particles present in the plasma of mice (8).LCAT is associated with transformation and remodeling of HDL. Plasma from LCAT-deficient patients (10, 11 ) contains small, discoidal HDL particles that probably represent nascent HDL (12). These particles undergo marked changes in composition and morphology when LCAT is added, suggesting an essential role of this enzyme in the biogenesis of circulating HDL.In Plasmid DNA was purified by alkaline hydrolysis (14) followed by two rounds of ultracentrifugation. Genomic DNA fragments were obtained by BamHI/NsiI or AatII/SspI digestion of clones pUCLCATBamNsi and pGEMAlbLCAT, respectively ( Fig. 1), separated from vector sequences by agarose gel electrophoresis, extracted from gels using the Geneclean kit (Bio 101, La Jolla, CA), and dialyzed in injection buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.4).Creation of transgenic mice. Methods utilized in the creation of transgenic mice have been previously described (15). Fertilized embryos used for the microinjection were derived from matings of inbred FVB mice (Charles River Laboratories, Wilmington, MA). The Hu apo AI and Hu apo All transgenic mice used in these studies have previously been described (16,17). These transgenic lines were created and maintained in the C57BL/6 background. In all the studies involving combinations of Hu apo Al, Hu apo All, and LCAT transgenes, the genetic background of each of the transgenic and control mice was (FVB x C57BL/6) F1 hybrids.Preparation and analysis ofDNA and RNA by Southern and Northern blot. Tail tip DNA from 3-wk-old mice was screened for integration of Hu LCAT gene sequences by PCR. Primers were directed to synthesize the exon 6 of the Hu LCAT gene. Amplification reaction using nontransgenic mouse DNA as a template yielded no amplification products. In some experiments, integration of the full-length genomic construct was detected by Southern blot hybridization. 10 jig genomic DNA was digested overnight with 5 U Pstl/jig DNA, electrophoresed in a 1% agarose gel, and transferred to a nylon membrane (Sigma Chemical Co., S. Louis, MO). DNA was cross-linked to the membranes (Stratagene, La Jolla, CA) and hybridized with a 32P-radiolabeled full-length Hu LCAT cDNA as a pro...

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