Quantitative determination of estradiol fatty acid esters in lipoprotein fractions in human blood

According to experimental studies, 17 beta-estradiol associates with lipoproteins in blood in the form of fatty acid esters. However, concentrations of endogenous estradiol esters in human lipoprotein fractions have not been previously reported. We investigated the distribution of estradiol fatty acid esters between plasma lipoproteins in 10 healthy women during late pregnancy. Following extraction from serum and ultracentrifugally isolated, gel-filtered lipoproteins, estradiol esters were separated from nonesterified estradiol by column chromatography. After saponification and chromatographic purification of the estradiol ester fraction, the concentration of hydrolyzed esters was determined by estradiol time-resolved fluoroimmunoassay. Of total serum estradiol, a mean of 0.7% (549 pmol/liter, n = 10) was in the form of fatty acid esters. Estradiol fatty acid ester concentrations measured in serum and lipoprotein fraction correlated positively (n = 10; r = 0.98; P < 0.001). The majority of lipoprotein estradiol esters, 54%, was recovered in high-density lipoprotein, and 28% in low-density lipoprotein fraction. Most lipoprotein fractions contained undetectable amounts of nonesterified estradiol (< 36 pmol/liter). In conclusion, our results indicate that estradiol fatty acid esters are mostly bound by lipoproteins in blood in vivo.     

ApoB-containing lipoproteins promote infectivity of chlamydial species in human hepatoma cell line

AIM: To evaluate the direct binding of two main chlamydial biovars (C. trachomatis and C. pneumoniae) to plasma lipoproteins and its effect on chlamydial infection rate in human hepatoma cell line (HepG2 cells).METHODS: Murine plasma lipoproteins were fractionated and isolated using fast-performance liquid chromatography (FPLC), spotted on nitrocellulose membrane and incubated with chlamydial suspensions. Direct binding of chlamydial particles to lipoprotein fractions has been studied using lipopolysaccharide-specific antibodies in immuno-dot blot binding assay and immunoprecipitation analysis. Immunostaining protocol as well as flow cytometry analysis have been employed to study the infectivity rate of chlamydial species in HepG2 cells.RESULTS: Elementary bodies of both C. trachomatis and C. pneumoniae bind ApoB-containing fractions of plasma lipoproteins. That binding becomes stronger when heat-denatured FPLC fractions are used, suggesting a primary role of apolipoproteins in interaction between chlamydial particle and lipoprotein. Both chlamydial biovars efficiently propagate in human hepatoma cell line - HepG2 cells even in serum free conditions forming late-stage inclusion bodies and releasing extracellular elementary bodies. Preincubation of C. trachomatis and C. pneumoniae with native ApoB-containing lipoproteins enhances the rate of chlamydial infection in HepG2 cells.CONCLUSION: A productive infection caused by C. trachomatis and C. pneumoniae may take place in human-derived hepatocytes revealing hepatic cells as possible target in chlamydial infection. Obtained results may suggest the participation of lipoprotein receptors in the mechanism of attachment and/or entry of chlamydial particles into target cells.     

Cultured normal human hepatocytes do not synthesize lipoprotein-associated coagulation inhibitor: evidence that endothelium is the principal site of its synthesis.

Human plasma contains a factor Xa-dependent inhibitor of tissue factor/factor VIIa complex termed lipoprotein-associated coagulation inhibitor (LACI). The present study examines the site(s) of LACI synthesis. In this study, cultured hepatocytes isolated from normal human liver were found to be essentially negative in LACI mRNA as revealed by Northern blot analysis using a full-length LACI cDNA as probe. The conditioned media from these cultures were also essentially negative for LACI activity. Similarly, poly(A)+ RNA obtained from normal human liver did not contain detectable LACI mRNA. In contrast, cultured human umbilical vein endothelial cells and human lung tissue (rich in endothelium) both contained abundant amounts of LACI mRNA. Moreover, erythrocyte lysates and culture media from normal monocytes, lymphocytes, or neutrophils did not contain measurable LACI activity; these cells were also negative for LACI mRNA. Platelets, however, contained LACI activity. The likely source of platelet LACI is the megakaryocyte cell since a megakaryocyte cell line (MEG-01) was found to contain LACI mRNA and to secrete small amounts of LACI activity. Additionally, human vascular smooth muscle cells and lung fibroblasts were also found to synthesize only small amounts of LACI. From these observations, we conclude that normal liver does not synthesize LACI and that endothelium is the principal source of plasma LACI. The undegraded LACI synthesized by endothelial cells had a molecular weight of approximately 41,000.     

New protein in human blood plasma, rich in proline, with lipid-binding properties.

A protein that binds to a lecithin-stabilized triglyceride emulsion has been separated from plasma after removal of major lipoprotein classes by ultracentrifugation at density 1.21 g/ml. This protein, rich in proline, has been purified to electrophoretic and immunochemical homogeneity by subsequent gel and ion-exchange chromatography. In native plasma and after purification, it exists as a large particle exceeding 10(6) daltons, but a single component with a molecular weight of about 74,000 is found upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Although the purified protein contains very little bound lipid and is not present in the major lipoprotein classes from post-absorptive individuals, it is present in chylomicrons. Its concentration in plasma varies from 12 to 41 mg/dl and is significantly correlated with that of cholesterol in lipoproteins of very low and low density but not in those of high denisty.     

Effect of massive obesity on low and high density lipoprotein binding to human adipocyte plasma membranes

Adipose tissue is a major cholesterol storage organ in man, and turnover of this slowly exchangeable pool is dependent on low and high density lipoproteins which deliver and remove cholesterol from this site. To determine whether lipoprotein binding is altered in the obese state, we examined the binding of low density lipoprotein (LDL) and high density lipoprotein (HDL2 and HDL3) to purified adipocyte plasma membranes obtained from omental fat depots of massively obese patients (BMI greater than 40 kg/m2) and lean subjects. The specific binding and uptake of 125I-HDL2 and 125I-HDL3 were greater for obese than for lean adipocytes. Scatchard analysis of binding studies using purified adipocyte plasma membranes and varying amounts of labeled HDL2 or HDL3 demonstrated a higher binding affinity (lower Kd) for HDL2 and higher binding capacity (Bmax) value for both HDL2 and HDL3 in obese as compared to lean. 125I-LDL specific binding was somewhat lower in obese than in lean membranes but this difference was not statistically significant. The cholesterol content of isolated omental adipocytes expressed on a cellular basis or as the cholesterol/triglyceride ratio (mg chol/g of lipid) were similar in the obese and lean subjects. Furthermore, 125I-LDL, 125I-HDL2 or 125I-HDL3 specific binding did not correlate with cellular cholesterol content or with cholesterol/triglyceride ratio. These findings indicate that lipoprotein binding to adipocytes is altered in obesity and is characterized by up-regulation of HDL (particularly HDL2) binding with little change in LDL binding. We conclude from this study that obesity has a profound effect on the expression of HDL binding sites in human adipocytes and that LDL and HDL binding in fat cells are regulated differently.     

The lipoprotein-associated coagulation inhibitor that inhibits the factor VII-tissue factor complex also inhibits factor Xa: insight into its possible mechanism of action

Blood coagulation is initiated when plasma factor VII(a) binds to its essential cofactor tissue factor (TF) and proteolytically activates factors X and IX. Progressive inhibition of TF activity occurs upon its addition to plasma. This process is reversible and requires the presence of VII(a), catalytically active Xa, Ca2+, and another component that appears to be associated with the lipoproteins in plasma, a lipoprotein-associated coagulation inhibitor (LACI). A protein, LACI(HG2), possessing the same inhibitory properties as LACI, has recently been isolated from the conditioned media of cultured human liver cells (HepG2). Rabbit antisera raised against a synthetic peptide based on the N-terminal sequence of LACI(HG2) and purified IgG from a rabbit immunized with intact LACI(HG2) inhibit the LACI activity in human serum. In a reaction mixture containing VIIa, Xa, Ca2+, and purified LACI(HG2), the apparent half-life (t1/2) for TF activity was 20 seconds. The presence of heparin accelerated the initial rate of inhibition threefold. Antithrombin III alpha alone had no effect, but antithrombin III alpha with heparin abrogated the TF inhibition. LACI(HG2) also inhibited Xa with an apparent t1/2 of 50 seconds. Heparin enhanced the rate of Xa inhibition 2.5-fold, whereas phospholipids and Ca2+ slowed the reaction 2.5-fold. Xa inhibition was demonstrable with both chromogenic substrate (S-2222) and bioassays, but no complex between Xa and LACI(HG2) could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Nondenaturing PAGE, however, showed that LACI(HG2) bound to Xa but not to X or Xa inactivated by diisopropyl fluorophosphate. Thus, LACI(HG2) appears to bind to Xa at or near its active site. Bovine factor Xa lacking its gamma-carboxyglutamic acid-containing domain, BXa(-GD), through treatment with alpha-chymotrypsin, was used to further investigate the Xa requirement for VIIa/TF inhibition by LACI(HG2). LACI(HG2) bound to BXa(-GD) and inhibited its catalytic activity against a small molecular substrate (Spectrozyme Xa), though at a rate approximately sevenfold slower than native BXa. Preincubation of LACI(HG2) with saturating concentrations of BXa(-GD) markedly retarded the subsequent inhibition of BXa. The VII(a)/TF complex was not inhibited by LACI(HG2) in the presence of BXa(-GD), and further, preincubation of LACI(HG2) with BXa(-GD) slowed the inhibition of VIIa/TF after the addition of native Xa. The results are consistent with the hypothesis that inhibition of VII(a)/TF involves the formation of a VIIa-TF-XA-LACI complex that requires the GD of XA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification of a galanin receptor from pig brain.

A galanin receptor protein was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) from pig brain membranes and then purified by single-step affinity chromatography. The product exhibits saturable and specific binding for galanin with a binding activity of 17 nmol/mg of protein and a dissociation constant (Kd) of 10 nM. This represents a 300,000-fold purification over the detergent-solubilized fraction with a final recovery of 31% of the initial membrane galanin binding activity. Gel electrophoresis of the affinity-purified material showed a single polypeptide of 54 kDa by silver staining and after radioiodination. Cross-linking of a purified fraction affinity-labeled with 125I-labeled galanin revealed a single band for the galanin-receptor complex at 57 kDa. The general binding characteristics of the purified preparation appeared to be identical to those of the crude soluble material as far as specificity toward galanin and the structural requirement for galanin are concerned. In contrast, unlike the CHAPS-soluble galanin receptor, binding of 125I-labeled galanin to the purified galanin receptor was not sensitive to guanine nucleotides, suggesting that dissociation of the inhibitory guanine nucleotide binding protein from the galanin receptor occurred during purification. The purification to homogeneity of a galanin receptor paves the way toward its sequencing and cloning.     

Purification and partial characterization of a receptor protein for mouse interferon gamma.

A receptor protein for mouse interferon gamma has been purified from solubilized plasma membranes of the mouse monomyelocytic cell line WEHI-3. Sequential wheat germ agglutinin and ligand affinity chromatography of membranes extracted with octyl beta-D-glucopyranoside resulted in at least a 680-fold purification of the receptor, as measured by precipitating it in association with liposomes composed of phosphatidylcholine. The purified receptor bound 125I-labeled recombinant mouse interferon gamma (rMuIFN-gamma) with a Kd of 10 nM, a value comparable to that obtained with isolated membranes (3.5 nM). PAGE analysis of radiolabeled (with either 35S or 125I) receptor preparations consistently revealed a major band of 95 kDa. This species was degraded with time to smaller fragments, principally one of 60 +/- 5 kDa. Treatment with peptide:N-glycosidase F reduced the apparent molecular masses of the proteins in the 95- and 60-kDa regions by 15-20 kDa each. GR-20, a monoclonal antibody against the receptor, completely inhibited specific binding of 125I-labeled rMuIFN-gamma to WEHI-3 cells, blocked the induction of priming by rMuIFN-gamma of macrophage-mediated tumor cell killing, removed binding activity for 125I-labeled rMuIFN-gamma from solubilized membranes, and immunoprecipitated a single 95-kDa protein from the extract of surface labeled (125I) WEHI-3 cells. Cross-linking of 125I-labeled rMuIFN-gamma to its receptor yielded a complex of 125 +/- 5 kDa, consistent with the binding of the dimeric form of mouse interferon gamma (32 kDa) to a membrane protein of 95 kDa. These data suggest that the receptor for mouse interferon gamma (or a ligand-binding subunit thereof) is a glycoprotein of 95 kDa.     

A physiologic role for the esterified cholesterol transfer protein: In vivo studies in rabbits and pigs

The observation that pig plasma is deficient in esterified cholesterol transfer activity has been exploited in an attempt to establish an in vivo role for the esterified cholesterol transfer protein. The plasma high density lipoproteins (HDL) of pigs and also of rabbits (a species known to posses an active esterified cholesterol transfer protein) were labeled with ³H in the esterified cholesterol moiety and with ¹²⁵I in the apoprotein moieties and reinjected into the respective species. In both rabbits and pigs, the removal of ¹²⁵I from the recipient HDL fraction was parallel to that from the whole plasma, with negligible ¹²⁵I appearing in other plasma lipoprotein fractions. In the pig, the removal of esterified ³H-cholesterol from the recipient HDL was very similar to that of ¹²⁵I; there was only minimal appearance in other lipoproteins. In the rabbit, however, there was a major in vivo transfer of esterified ³H-cholesterol from HDL to other fractions. It has been concluded that an active esterified cholesterol transfer protein is probably necessary to achieve the in vivo transfer of esterified cholesterol from HDL to other plasma lipoproteins.     

The role of lipoproteins and receptor-mediated endocytosis in the transport of bacterial lipopolysaccharide

The addition of bacterial lipopolysaccharide (LPS) from Escherichia coli 0111:B4 to human monocyte-macrophages cultured in serum results in suppression of scavenger receptor activity. The present studies were performed to examine if the effect on scavenger receptor activity was mediated by LPS alone or by LPS in association with lipoproteins. Radioiodinated LPS (125I-LPS) was added to human plasma in vitro and to normal and hyperlipidemic rabbit plasma in vitro and in vivo to determine the distribution of 125I-LPS among the lipoprotein classes. It was found that all lipoprotein classes bound LPS in direct proportion to their plasma cholesterol concentration. LPS alone was compared to LPS bound to low density lipoprotein (LDL), high density lipoprotein, or reductively-methylated LDL for their abilities to suppress scavenger receptor activity in monocyte-macrophages in lipoprotein-free serum. Only LPS bound to LDL (LPS-LDL) demonstrated an effect similar to that observed when LPS was added to cells in serum. Either unlabeled LDL or unlabeled LPS-LDL complexes competed with the uptake of 125I-LPS-LDL complexes, which appeared to proceed by receptor-mediated endocytosis. In contrast to the uptake of 125I-LDL, the uptake of 125I-LPS-LDL by cultured monocyte-macrophages was not followed by its hydrolysis and the release of its radioactive degradation products into the medium. The association of LPS with lipoproteins was very stable and appeared to be mediated by a lipid-lipid interaction. We hypothesize that LPS bound to lipoproteins may be transported into the artery wall and may initiate the atherosclerotic reaction.     

Alpha 2-macroglobulin binds and inhibits activated protein C.

In previous studies using a nonhuman primate model of Protein C (PC) activation in vivo, immunoblotting showed substantial amounts of activated PC (APC) in a high molecular weight complex with what was presumed to be a previously unrecognized APC binding protein. This APC complex can also be formed in citrated plasma in vitro. It is of low electrophoretic mobility, sodium dodecyl sulfate (SDS) stable, with an apparent Mr of 320 Kd. Its purification from human plasma was accomplished using barium citrate adsorption, sequential polyethylene glycol (PEG) precipitations, diethylaminoethyl sepharose chromatography, AcA-34 gel filtration, and zinc-chelate affinity chromatography. This was monitored by subjecting the fractions to nondenaturing polyacrylamide gel electrophoresis (PAGE), transfer to polyvinylidene-difluoride membranes, and probing with 125I-labeled human APC. The purified APC-binding protein was homogeneous by SDS-PAGE with an Mr of 275 Kd. Its identity as alpha 2-macroglobulin (alpha 2M) was demonstrated immunochemically. Complex formation between alpha 2M and APC was found to be almost completely inhibited by EDTA, but to a lesser extent by citrate. Complex formation could also be prevented by active site inhibition with D-Phenylalanyl-L-Prolyl-L-Arginine-Chloromethyl Ketone (PPACK) or pretreatment of alpha 2M with methylamine. Incubation of APC (33 nmol/L) with alpha 2M (1 mumol/L) resulted in time-dependent inhibition of APC anticoagulant activity when measured using an activated partial thromboplastin time based APC assay. These data show that alpha 2M binds and inhibits APC in vitro and the interaction is both metal-ion and active-site dependent, requiring functionally intact alpha 2M. As the complexes formed in vitro comigrate electrophoretically with those observed in vivo after PC activation, it is suggested that alpha 2M is a physiologically relevant inhibitor involved in the processing of APC in vivo.     

Isolation of the human plasma corticotrophin-releasing factor-binding protein

We report the purification of human corticotrophin-releasing factor-binding protein (hCRF-BP) using repeated affinity chromatography (with the aid of synthetic CRF immobilized on Sepharose 4B solid phase) followed by gel filtration. Presence of the binding protein was tracked throughout the procedure by its ability to inhibit binding of 125I-labelled hCRF to an hCRF antiserum; normal human plasma exhibits 80% inhibition in this system whereas sheep plasma, which does not contain an hCRF-BP, has no effect. Affinity cross-linking of 125I-labelled hCRF to the purified hCRF-BP was performed using disuccinimidyl suberate (1 mmol/l). SDS electrophoresis of the purified CRF cross-linked binding protein followed by radioautography resulted in one major band of Mr 37,000 which corresponded to our original molecular weight estimate based on the elution position of the binding protein on Sephacryl S-200. A 10(7)-fold purification of the hCRF-BP resulted in a preparation estimated to be 95% pure, with an overall yield of 5%.     

Three types of naturally occurring modified lipoproteins induce intracellular lipid accumulation due to lipoprotein aggregation.

Low density lipoprotein (LDL) from patients with coronary atherosclerosis and diabetes mellitus as well as in vitro desialylated LDL, glycosylated LDL, and lipoprotein (a) caused a twofold to fourfold rise in cholesteryl ester in cultured human blood monocytes and intimal smooth muscle cells isolated from normal aorta. Native LDL from healthy subjects failed to induce intracellular lipid accumulation. We have demonstrated by laser correlative photometry and gel filtration chromatography that in vivo and in vitro modified lipoproteins form aggregates under cell culture conditions. The degree of modified lipoprotein aggregation directly correlated with the ability of these lipoproteins to elevate the cholesteryl ester content of cultured cells. Modified lipoprotein aggregates isolated by gel filtration induced a threefold to fivefold elevation in cellular cholesteryl ester content. Aggregates of 125I-modified LDL were taken up and degraded fivefold to sevenfold more effectively as compared with nonaggregated lipoproteins. The uptake and degradation of 125I-labeled aggregates were strongly inhibited by unlabeled aggregates, latex beads, and cytochalasin B but not by native or acetylated LDL. These data indicate that uptake of lipoprotein aggregates occurred by phagocytosis. Obtained results suggest that modified lipoprotein aggregation may be the key condition for lipid accumulation.     

Purification and subunit structure of a putative K+-channel protein identified by its binding properties for dendrotoxin I.

The binding protein for the K+-channel toxin dendrotoxin I was purified from a detergent extract of rat brain membranes. The purification procedure utilized chromatography on DEAE-Trisacryl, affinity chromatography on a dendrotoxin-I-Aca 22 column, and wheat germ agglutinin-Affigel 10 with a final 3800- to 4600-fold enrichment and a recovery of 8-16%. The high affinity (Kd, 40-100 pM) and specificity of the binding site are retained throughout the purification procedure. Analysis of the purified material on silver-stained NaDodSO4/polyacrylamide gel revealed three bands of Mr 76,000-80,000, 38,000, and 35,000. Interestingly, the binding site for 125I-labeled mast cell degranulating peptide, another putative K+-channel ligand from bee venom, which induces long-term potentiation in hippocampus, seems to reside on the same protein complex, as both binding sites copurify through the entire purification protocol.     

Separation of the insulin-like growth factor-binding proteins in plasma and purification of the larger molecular weight species

Methods were developed for purification of the high mol wt (150K) insulin-like growth factor (IGF)-binding protein and its acid stable (70K) component from human plasma. High mol wt IGF-binding protein was highly purified by chromatography of Cohn IV-1 fraction of human plasma on Concanavalin A-Sepharose followed by chromatography on IGF-I-Sepharose. The acid-stable component of the high mol wt IGF-binding protein was purified to near homogeneity by chromatography of Cohn IV-1 fraction of human plasma on Con-A Sepharose, followed by chromatography on Sephadex G-50 at pH 2.5 and subsequent chromatography on IGF-I-Sepharose. Both fractions obtained after IGF-I-Sepharose chromatography were capable of binding [125I]IGF-I and gave a single protein band of 79,000 mol wt, when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Coomassie blue staining), suggesting that the large mol wt species may be a dimer of identical or iso-mol wt subunits. Three minor contaminants of less than 5% each were detected upon subsequent silver staining. These methods represent important tools that should aid in furthering our understanding of the role of the IGF carrier proteins in the actions of IGF-I and IGF-II.     

Plasma antigen levels of the lipoprotein-associated coagulation inhibitor in patient samples

Human plasma contains an inhibitor of tissue factor-initiated coagulation known as the lipoprotein-associated coagulation inhibitor (LACI) or also known as the extrinsic pathway inhibitor (EPI). A competitive fluorescent immunoassay was developed to measure the plasma concentration of LACI in samples from normal individuals and patients with a variety of diseases. The LACI concentration in an adult control population varied from 60% to 160% of the mean with a mean value corresponding to 89 ng/mL or 2.25 nmol/L. Plasma LACI levels were not decreased in patients with severe chronic hepatic failure, warfarin therapy, primary pulmonary hypertension, thrombosis, or the lupus anticoagulant. Plasma LACI antigen was decreased in some, but not all patients with gram-negative bacteremia and evidence for disseminated intravascular coagulation. Plasma LACI levels were elevated in women undergoing the early stages of labor (29%), in patients receiving intravenous tissue-type plasminogen activator (45%), and in patients receiving intravenous heparin (375%). A radioligand blot of the pre- and post-heparin plasma samples shows the increase to be in a 40-Kd form of LACI. Very low levels of plasma LACI antigen were found in patients with homozygous abetalipoproteinemia and hypobetalipoproteinemia, diseases associated with low plasma levels of apolipoprotein B containing lipoproteins. Following the injection of heparin into one patient with homozygous abetalipoproteinemia, the plasma LACI antigen level increased to a level comparable with that in normal individuals after heparin treatment.     

Heparan sulfate in perlecan promotes mouse atherosclerosis: roles in lipid permeability, lipid retention, and smooth muscle cell proliferation

Heparan sulfate (HS) has been proposed to be antiatherogenic through inhibition of lipoprotein retention, inflammation, and smooth muscle cell proliferation. Perlecan is the predominant HS proteoglycan in the artery wall. Here, we investigated the role of perlecan HS chains using apoE null (ApoE0) mice that were cross-bred with mice expressing HS-deficient perlecan (Hspg2(Delta3/Delta3)). Morphometry of cross-sections from aortic roots and en face preparations of whole aortas revealed a significant decrease in lesion formation in ApoE0/Hspg2(Delta3/Delta3) mice at both 15 and 33 weeks. In vitro, binding of labeled mouse triglyceride-rich lipoproteins and human LDL to total extracellular matrix, as well as to purified proteoglycans, prepared from ApoE0/Hspg2(Delta3/Delta3) smooth muscle cells was reduced. In vivo, at 20 minutes influx of human (125)I-LDL or mouse triglyceride-rich lipoproteins into the aortic wall was increased in ApoE0/Hspg2(Delta3/Delta3) mice compared to ApoE0 mice. However, at 72 hours accumulation of (125)I-LDL was similar in ApoE0/Hspg2(Delta3/Delta3) and ApoE0 mice. Immunohistochemistry of lesions from ApoE0/Hspg2(Delta3/Delta3) mice showed decreased staining for apoB and increased smooth muscle alpha-actin content, whereas accumulation of CD68-positive inflammatory cells was unchanged. We conclude that the perlecan HS chains are proatherogenic in mice, possibly through increased lipoprotein retention, altered vascular permeability, or other mechanisms. The ability of HS to inhibit smooth muscle cell growth may also influence development as well as instability of lesions.     

Desialylation of human apolipoprotein E decreases its binding to human high-density lipoprotein and its ability to deliver esterified cholesterol to the liver.

Apolipoprotein E (apoE) plays a significant role in the delivery of high-density lipoprotein (HDL) cholesterol to the liver via the apoB/E receptor. The roles of the apoE sialylation status in its association with HDL and in the reverse cholesterol transport (RCT) function of HDL have not been well defined. Furthermore, long-term ethanol treatment impairs apoE sialylation and leads to its decreased content in HDL. Therefore, we investigated the association of either sialo apoE (SapoE) or desialo apoE (DSapoE) with HDL and its effect on the RCT function of HDL. The dextran sulfate precipitation method showed that [125I]DSapoE binding to HDL was 27.3% (P < .02) to 35.5% (P < .001) lower versus [125I]SapoE. Scatchard analysis of the specific binding data showed that [125I]SapoE had 11.2 times more affinity for HDL than [125I]DSapoE based on size-exclusion chromatography (Kd = 89.7 v 1,010 nmol/L). Similarly, [1251]HDL had 4.5 times more affinity for SapoE compared with DSapoE based on solid-phase binding (Kd = 21.9 v 104.4 nmol/L). Furthermore, esterified cholesterol uptake from reconstituted HDL particles (rHDLs) by HepG2 cells increased over basal uptake up to 153% when rHDLs contained SapoE, versus only 37% with DSapoE. Enzymatic resialylation of DSapoE completely restored its HDL-binding and RCT properties, identical to those of SapoE. It is therefore concluded that desialylation of apoE decreases its binding to plasma HDL, leading to an impaired RCT function.     

Platelet lipoprotein interplay: trigger of foam cell formation and driver of atherosclerosis

In the last decade, it was recognized that platelets and lipoproteins play a pivotal role in both early and late atherogenesis. Beside cellular interactions of platelets with other blood cells and vascular cells, interactions with lipoproteins seem to be quite important. Lipoproteins are fundamental 'players' in atherogenesis since they change the properties of different cells involved in atherosclerosis and thrombosis. Several studies have already shown that low density lipoproteins (LDL) are involved in the initiation of platelet signalling pathways. Platelets of hypercholesterolemic patients show hyperaggregability in vitro and enhanced activity in vivo. This review elucidates the major aspects concerning how native and modified lipoproteins influence the activation and metabolic behaviour of platelets, and shows a new way by which platelet-mediated lipoprotein transfer might contribute to foam cell formation. In hyperlipidaemia, circulating platelets are activated. This is accompanied by increased platelet aggregation, platelet-leukocyte aggregate formation, and platelet-induced superoxide anion production. Furthermore, oxidized LDL induces monocyte adhesion to the endothelium, migration and proliferation of smooth muscle cells, injures cells, interferes with nitric oxide release, and promotes procoagulant properties of vascular cells. New data about platelet-mediated lipoprotein transport and consequent foam cell formation, however, provide proof of how platelets might contribute to atheromatous lesion formation.     

Regulation of low density lipoprotein receptors by plasma lipoproteins from patients with abetalipoproteinemia.

Despite an absence of low density lipoproteins (LDLs) and chylomicron remnants from plasma, the rates of cholesterol synthesis or the number of LDL receptors expressed on freshly isolated cells from patients with abetalipoproteinemia are not markedly increased. These observations suggest that other lipoprotein particles present in the plasma of patients with abetalipoproteinemia may regulate LDL receptor activity and the rates of cellular cholesterol synthesis in this disorder. In the present report we have studied the effects of lipoprotein fractions from the plasma of normal subjects, patients with abetalipoproteinemia, and a patient with dysbetalipoproteinemia on the binding, internalization, and degradation of 125I-labeled LDL (125I-LDL) by cultured human fibroblasts. LDL from normal subjects or the high density lipoprotein fraction HDL2 from the plasma of patients with abetalipoproteinemia effectively down-regulated LDL receptor activity (greater than 50% inhibition at 20 micrograms of protein per ml). HDL2 from the plasma of patients with abetalipoproteinemia also effectively reduced the binding, internalization, and degradation of 125I-LDL by cultured human fibroblasts. 125I-HDL2 from the plasma of patients with abetalipoproteinemia was bound, internalized, and degraded by cultured human fibroblasts; this process was competitively inhibited by unlabeled normal LDL or HDL2 from abetalipoproteinemic plasma and was 1/6th to 1/8th times as high when 125I-HDL2 was incubated with fibroblasts from a patient with receptor-negative homozygous familial hypercholesterolemia. We conclude that lipoproteins present in the HDL2 fraction of plasma from patients with abetalipoproteinemia (which are relatively rich in apoprotein E) are effective regulators of LDL receptor activity in normal human fibroblasts. These in vitro findings may explain why the in vivo rates of cholesterol synthesis and the number of LDL receptors expressed on freshly isolated cells from patients with abetalipoproteinemia are not markedly increased.