Negative regulation of human fibrinogen gene expression by peroxisome proliferator-activated receptor alpha agonists via inhibition of CCAAT box/enhancer-binding protein beta.

Fibrinogen is a coagulation factor and an acute phase reactant up-regulated by inflammatory cytokines, such as interleukin 6 (IL-6). Elevated plasma fibrinogen levels are associated with coronary heart diseases. Fibrates are clinically used hypolipidemic drugs that act via the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR alpha). In addition, most fibrates also reduce plasma fibrinogen levels, but the molecular mechanism is unknown. In this study, we demonstrate that fibrates decrease basal and IL-6-stimulated expression of the human fibrinogen-beta gene in human primary hepatocytes and hepatoma HepG2 cells. Fibrates diminish basal and IL-6-induced fibrinogen-beta promoter activity, and this effect is enhanced in the presence of co-transfected PPAR alpha. Site-directed mutagenesis experiments demonstrate that PPAR alpha activators decrease human fibrinogen-beta promoter activity via the CCAAT box/enhancer-binding protein (C/EBP) response element. Co-transfection of the transcriptional intermediary factor glucocorticoid receptor-interacting protein 1/transcriptional intermediary factor 2 (GRIP1/TIF2) enhances fibrinogen-beta gene transcription and alleviates the repressive effect of PPAR alpha. Co-immunoprecipitation experiments demonstrate that PPAR alpha and GRIP1/TIF2 physically interact in vivo in human liver. These data demonstrate that PPAR alpha agonists repress human fibrinogen gene expression by interference with the C/EBP beta pathway through titration of the coactivator GRIP1/TIF2. We observed that the anti-inflammatory action of PPAR alpha is not restricted to fibrinogen but also applies to other acute phase genes containing a C/EBP response element; it also occurs under conditions in which the stimulating action of IL-6 is potentiated by dexamethasone. These findings identify a novel molecular mechanism of negative gene regulation by PPAR alpha and reveal the direct implication of PPAR alpha in the modulation of the inflammatory gene response in the liver.

The nuclear receptors peroxisome proliferator-activated receptor alpha and Rev-erbalpha mediate the species-specific regulation of apolipoprotein A-I expression by fibrates.

Fibrates are widely used hypolipidemic drugs which activate the nuclear peroxisome proliferator-activated receptor (PPAR) alpha and thereby alter the transcription of genes controlling lipoprotein metabolism. Fibrates influence plasma high density lipoprotein and its major protein, apolipoprotein (apo) A-I, in an opposite manner in man (increase) versus rodents (decrease). In the present study we studied the molecular mechanisms of this species-specific regulation of apoA-I expression by fibrates. In primary rat and human hepatocytes fenofibric acid, respectively, decreased and increased apoA-I mRNA levels. The absence of induction of rat apoA-I gene expression by fibrates is due to 3 nucleotide differences between the rat and the human apoA-I promoter A site, rendering a positive PPAR-response element in the human apoA-I promoter nonfunctional in rats. In contrast, rat, but not human, apoA-I transcription is repressed by the nuclear receptor Rev-erbalpha, which binds to a negative response element adjacent to the TATA box of the rat apoA-I promoter. In rats fibrates increase liver Rev-erbalpha mRNA levels >10-fold. In conclusion, the opposite regulation of rat and human apoA-I gene expression by fibrates is linked to differences in cis-elements in their respective promoters leading to repression by Rev-erbalpha of rat apoA-I and activation by PPARalpha of human apoA-I. Finally, Rev-erbalpha is identified as a novel fibrate target gene, suggesting a role for this nuclear receptor in lipid and lipoprotein metabolism.

Retinoids increase human apo C-III expression at the transcriptional level via the retinoid X receptor. Contribution to the hypertriglyceridemic action of retinoids.

Hypertriglyceridemia is a metabolic complication of retinoid therapy. In this study, we analyzed whether retinoids increase the expression of apo C-III, an antagonist of plasma triglyceride catabolism. In men, isotretinoin treatment (80 mg/d; 5 d) resulted in elevated plasma apo C-III, but not apo E concentrations. In human hepatoma HepG2 cells, retinoids increased apo C-III mRNA and protein production. Transient transfection experiments indicated that retinoids increase apo C-III expression at the transcriptional level. This increased apo C-III transcription is mediated by the retinoid X receptor (RXR), since LG1069 (4-[1-(5,6,7,8-tetrahydro-3,5,5,8, 8-pentamethyl-2-naphtalenyl)ethenyl]benzoic acid), a RXR-specific agonist, but not TTNPB ((E)- 4-[2-(5,6,7,8-tetrahydro-5,5,8, 8-tetramethyl-2-naphtalenyl)propenyl]benzoic acid), a retinoic acid receptor (RAR)-specific agonist, induced apo C-III mRNA in HepG2 cells and primary human hepatocytes. Mutagenesis experiments localized the retinoid responsiveness to a cis-element consisting of two imperfect AGGTCA sequences spaced by one oligonucleotide (DR-1), within the previously identified C3P footprint site. Cotransfection assays showed that RXR, but not RAR, activates apo C-III transcription through this element either as a homo- or as a heterodimer with the peroxisome proliferator-activated receptor. Thus, apo C-III is a target gene for retinoids acting via RXR. Increased apo C-III expression may contribute to the hypertriglyceridemia and atherogenic lipoprotein profile observed after retinoid therapy.

Transcriptional regulation of apolipoprotein A-I gene expression by the nuclear receptor RORalpha.

Since elevated concentrations of plasma high density lipoprotein (HDL) and its major apolipoprotein (apo), apoA-I, confer protection against atherosclerosis, considerable research efforts have focussed on the identification of factors regulating apoA-I gene expression in an attempt to increase its production. Nuclear receptors are interesting candidates because they are transcription factors whose activity is ligand-dependent. In the present study we identified the orphan receptor RORalpha1 as an activator of apoA-I gene transcription. In apoA-I-expressing intestinal Caco-2 cells, overexpression of the RORalpha1, but not the RORalpha2 or RORalpha3 isoforms, increased rat apoA-I gene transcription. Deletion and site-directed mutagenesis experiments identified a functional ROR-responsive element (RORE) in the rat and mouse apoA-I gene promoters, which overlaps with the TATA box. Gel shift experiments indicated that this RORE binds the RORalpha1 isoform, but not the RORalpha2 or RORalpha3 isoforms. Furthermore, compared with wild type mice, apoA-I mRNA levels were significantly lower in small intestines of staggerer mice homozygous for a deletion in the RORalpha gene. In addition, reverse transcriptase-polymerase chain reaction analysis revealed the expression of RORalpha in small intestinal epithelium and in Caco-2 cells. These data indicate a novel, physiological role for RORalpha1 in the regulation of genes involved in lipid and lipoprotein metabolism and possibly in the development of metabolic diseases, such as atherosclerosis.

Identification of a second promoter in the human c-ets-2 proto-oncogene.

We localized and characterized a new regulatory element with promoter activity in the human c-ets-2 intron 1. This promoter governs the expression of 5' divergent c-ets-2 transcripts through multiple start sites dispersed within 300 bp. Among the multiple start sites detected, three are major transcriptional initiation points. We detected transcripts initiated from this new promoter in various cell lines such as COLO 320, NBE, or HepG2 cells. This promoter exhibits transcriptional activity when linked to the CAT gene, and deletion constructs reveal that it contains activating and repressing elements. The sequence of the promoter reveals putative binding sites for ETS, MYB, GATA, and Oct factors. In addition, we show that this promoter is functionally conserved in the chicken.

Retinoids increase human apolipoprotein A-11 expression through activation of the retinoid X receptor but not the retinoic acid receptor.

Considering the link between plasma high-density lipoprotein (HDL) cholesterol levels and a protective effect against coronary artery disease as well as the suggested beneficial effects of retinoids on the production of the major HDL apolipoprotein (apo), apo A-I, the goal of this study was to analyze the influence of retinoids on the expression of apo A-II, the other major HDL protein. Retinoic acid (RA) derivatives have a direct effect on hepatic apo A-II production, since all-trans (at) RA induces apo A-II mRNA levels and apo A-II secretion in primary cultures of human hepatocytes. In the HepG2 human hepatoblastoma cell line, both at-RA and 9-cis RA as well as the retinoid X receptor (RXR)-specific agonist LGD 1069, but not the RA receptor (RAR) agonist ethyl-p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-l-pro penyl]-benzoic acid (TTNPB), induce apo A-II mRNA levels. Transient-transfection experiments with a reporter construct driven by the human apo A-II gene promoter indicated that 9-cis RA and at-RA, as well as the RXR agonists LGD 1069 and LG 100268, induced apo A-II gene expression at the transcriptional level. Only minimal effects of the RAR agonist TTNPB were observed on the apo A-II promoter reporter construct. Unilateral deletions and site-directed mutagenesis identified the J site of the apo A-II promoter mediating the responsiveness to RA. This element contains two imperfect half-sites spaced by 1 oligonucleotide. Cotransfection assays in combination with the use of RXR or RAR agonists showed that RXR but not RAR transactivates the apo A-II promoter through this element. By contrast, RAR inhibits the inductive effects of RXR on the apo A-II J site in a dose-dependent fashion. Gel retardation assays demonstrated that RXR homodimers bind, although with a lower affinity than RAR-RXR heterodimers, to the AH-RXR response element. In conclusion, retinoids induce hepatic apo A-II production at the transcriptional level via the interaction of RXR with an element in the J site containing two imperfect half-sites spaced by 1 oligonucleotide, thereby demonstrating an important role of RXR in controlling human lipoprotein metabolism. Since the J site also confers responsiveness of the apo A-II gene to fibrates and fatty acids via the activation of peroxisome proliferator-activated receptor-RXR heterodimers, this site can be considered a plurimetabolic response element.

Transcriptional induction of rat liver apolipoprotein A-I gene expression by glucocorticoids requires the glucocorticoid receptor and a labile cell-specific protein.

Treatment with glucocorticoids increases the concentration of plasma high-density lipoprotein (HDL), which is inversely correlated to the development of atherosclerosis. Previously, we demonstrated that repeated administration of glucocorticoids increases apolipoprotein (apo) A-I gene expression and decreases apoA-II gene expression in rat liver. In the present study, the mechanism of glucocorticoid action on hepatic apoA-I and apoA-II expression was studied. A single injection of rats with dexamethasone increased hepatic apoA-I mRNA levels within 6 h and further increases were observed after 12 h and 24 h. In contrast, liver apoA-II mRNA levels gradually decreased after dexamethasone treatment to less than 25% control levels after 24 h. In rat primary hepatocytes and McARH8994 hepatoma cells, addition of dexamethasone increased apoA-I mRNA levels in a time-dependent and dose-dependent manner, whereas apoA-II mRNA levels were unchanged. Simultaneous addition of the glucocorticoid antagonist RU486 prevented the increase in apoA-I mRNA levels after dexamethasone treatment, which suggests that the effects of dexamethasone are mediated through the glucocorticoid receptor. Inhibition of transcription by actinomycin D and nuclear-run-on experiments in McARH8994 cells and primary hepatocytes showed that dexamethasone induced apoA-I, but not apoA-II, gene transcription. Transient-transfection assays in McARH8994 cells with a chloramphenicol acetyl transferase vector driven by the rat-apoA-I-gene promoter demonstrated that the proximal apoA-I promoter could be induced by dexamethasone, and this effect could be abolished by simultaneous treatment with RU486. However, in COS-1 cells, apoA-I promoter transcription was not induced by dexamethasone or cotransfected glucocorticoid receptor. In addition, the induction of apoA-I gene transcription by dexamethasone was blocked by the protein-synthesis inhibitor cycloheximide, which suggests the presence of a labile protein involved in apoA-I gene activation by dexamethasone. In conclusion, our results demonstrate that dexamethasone regulates rat apoA-I, but not apoA-II, gene expression through direct action on the hepatocyte. The induction of apoA-I gene transcription by dexamethasone requires the glucocorticoid receptor and a labile cell-specific protein.

Fibrates increase human apolipoprotein A-II expression through activation of the peroxisome proliferator-activated receptor.

In view of the evidence linking plasma high density lipoprotein (HDL)-cholesterol levels to a protective effect against coronary artery disease and the widespread use of fibrates in the treatment of hyperlipidemia, the goal of this study was to analyze the influence of fibrates on the expression of apolipoprotein (apo) A-II, a major protein constituent of HDL. Administration of fenofibrate (300 mg/d) to 16 patients with coronary artery disease resulted in a marked increase in plasma apo A-II concentrations (0.34 +/- 0.11 to 0.45 +/- 0.17 grams/liter; P < 0.01). This increase in plasma apo A-II was due to a direct effect on hepatic apo A-II production, since fenofibric acid induced apo A-II mRNA levels to 450 and 250% of control levels in primary cultures of human hepatocytes and in human hepatoblastoma HepG2 cells respectively. The induction in apo A-II mRNA levels was followed by an increase in apo A-II secretion in both cell culture systems. Transient transfection experiments of a reporter construct driven by the human apo A-II gene promoter indicated that fenofibrate induced apo A-II gene expression at the transcriptional level. Furthermore, several other peroxisome proliferators, such as the fibrate, Wy-14643, and the fatty acid, eicosatetraynoic acid (ETYA), also induced apo A-II gene transcription. Unilateral deletions and site-directed mutagenesis identified a sequence element located in the J-site of the apo A-II promoter mediating the responsiveness to fibrates and fatty acids. This element contains two imperfect half sites spaced by 1 oligonucleotide similar to a peroxisome proliferator responsive element (PPRE). Cotransfection assays showed that the peroxisome proliferator activated receptor (PPAR) transactivates the apo A-II promoter through this AII-PPRE. Gel retardation assays demonstrated that PPAR binds to the AII-PPRE with an affinity comparable to its binding affinity to the acyl coA oxidase (ACO)-PPRE. In conclusion, in humans fibrates increase plasma apo A-II concentrations by inducing hepatic apo A-II production. Apo A-II expression is regulated at the transcriptional level by fibrates and fatty acids via the interaction of PPAR with the AII-PPRE, thereby demonstrating the pivotal role of PPAR in controlling human lipoprotein metabolism.

Fibrates downregulate apolipoprotein C-III expression independent of induction of peroxisomal acyl coenzyme A oxidase. A potential mechanism for the hypolipidemic action of fibrates.

Epidemiological and transgenic animal studies have implicated apo C-III as a major determinant of plasma triglyceride metabolism. Since fibrates are very efficient in lowering triglycerides, it was investigated whether fibrates regulate apo C-III gene expression. Different fibrates lowered rat liver apo C-III mRNA levels up to 90% in a dose- and time-dependent manner, whereas intestinal apo C-III mRNA remained constant. This decrease in liver apo C-III mRNA was rapid (1 d) and reversible, since it was restored to control levels within 1 wk after cessation of treatment. In addition, fenofibrate treatment abolished the developmental rise of hepatic apo C-III mRNA observed during the suckling-weaning period. Administration of fibrates to rats induced liver and intestinal expression of the acyl CoA oxidase gene, the rate-limiting enzyme for peroxisomal beta-oxidation of fatty acids. In primary cultures of rat and human hepatocytes, fenofibric acid lowered apo C-III mRNA in a time- and dose-dependent manner. This reduction in apo C-III mRNA levels was accompanied by a decreased secretion of apo C-III in the culture medium of human hepatocytes. In rat hepatocytes fenofibric acid induced acyl CoA oxidase gene expression, whereas acyl CoA oxidase mRNA remained unchanged in human hepatocytes. Nuclear run-on and transient transfection experiments of a reporter construct driven by the human apo C-III gene promoter indicated that fibrates downregulate apo C-III gene expression at the transcriptional level. In conclusion, these studies demonstrate that fibrates decrease rat and human liver apo C-III gene expression. In humans the mechanisms appears to be independent of the induction of peroxisomal enzymes. This downregulation of liver apo C-III gene expression by fibrates may contribute to the hypotriglyceridemic action of these drugs.

Negative regulation of the human apolipoprotein A-I promoter by fibrates can be attenuated by the interaction of the peroxisome proliferator-activated receptor with its response element.

Fibrates have been reported to modulate plasma high density lipoprotein cholesterol and apolipoprotein (apo) A-I concentrations. Therefore, the molecular mechanisms underlying the regulation of human apoA-I gene expression by fibrates was investigated. Fenofibrate reduced the expression of a reporter gene driven by the DNA sequences between -192 and +91 (BC-P-chloramphenicol acetyltransferase; CAT) relative to the apoA-I gene transcription start site approximately 3-fold. The sequences involved in the down-regulation of apoA-I gene transcription by fenofibrate were localized between -41 and +91 (P-CAT) relative to the transcription start site. The reduction of the expression of BC-P-CAT was dose-dependent and maximal at 500 microM (20 +/- 7%). Different peroxisome proliferators showed different levels of repression varying from 39 +/- 4% for fenofibrate, 43 +/- 5% for tetradecylthioacetic acid, 48 +/- 4% for bezafibrate, 54 +/- 2% for 5,8,11,14-eicotetraynoic acid, 76 +/- 2% for ciprofibrate, whereas Wy 14643 only marginally inhibited the expression of BC-P-CAT. By contrast, inclusion of sequences between -256 and -192 (ABC-P-CAT) attenuated the repression by fenofibrate. Furthermore, the apoA-IA site (-214 to -192; Awt-P-CAT) could counteract the repression of P-CAT by fenofibrate in the presence of cotransfected mPPAR alpha (peroxisome proliferator-activated receptor). In addition, the acyl-CoA oxidase-peroxisome proliferator response element (PPRE) could substitute the wild-type A-site in blocking the fenofibrate-induced reduction of the apoA-I promoter by mPPAR alpha. The protective effect of PPAR on fenofibrate induced inhibition of apoA-I expression was abolished after mutation of the direct repeat in the A site (Am-P-CAT). Consistent with these functional data only the wild-type, but not the mutated A site bound PPAR/retinoic X receptor heterodimers in gel shift assays. These data suggest that certain peroxisome proliferators can reduce the expression of the apoA-I promoter in a PPAR-independent fashion, through modulation of factors interacting with sequences localized between -41 and +91 of the apoA-I gene transcription initiation site. This inhibitory effect can be overcome when PPAR interacts with a functional PPRE, such as the apoA-I A site or the acyl-CoA oxidase-PPRE.

Transcription of the human apolipoprotein A-II is down-regulated by the first intron of its gene.

Several reports indicate that apoA-II, the second most abundant HDL protein, plays a crucial role in modulating the anti-atherogenic behavior of HDL. Regulatory elements located 5' to the human apoA-II promoter have been previously described. In this paper we report that the first intron of the human apoA-II gene down-regulates its own promoter and the ubiquitous thymidine kinase promoter both in HepG2 and Caco-2 cells. The intron contains three sequences which bind nuclear proteins, thus demonstrating the presence of regulatory elements downstream of the transcription start site of the apoA-II gene.

Apolipoprotein B polymorphism and altered apolipoprotein B concentrations in Congolese blacks.

The immunoreactivity of apolipoprotein B (apo B) in plasma obtained from 238 unrelated black African male subjects from the People's Republic of Congo was analysed by non-competitive Enzyme Linked-Immunosorbent Assay (ELISA) with monoclonal BIP 45 anti-LDL antibody. The polymorphism detected by BIP 45 monoclonal antibody is identical to the Ag(c,g) polymorphism. Antibody BIP 45 distinguishes three apo B allotypes (immunophenotypes) encoded by the two allelic genes apo B Ag(c) and apo B Ag(g). Because of co-dominant transmission, genotypes may be inferred from allotypes, and it has been shown that BIP 45 binds strongly to the Ag(c) factor and only weakly to the allelic Ag(g) factor. Analysis of the Congolese plasma samples indicated that 67.65% of them bound BIP 45 with low affinity (Ag(c-,g+) genotype), 28.15% with intermediate affinity (Ag(c+,g+) genotype) and 4.20% with high affinity (Ag(c+,g-) genotype). According to the Hardy-Weinberg equilibrium, this corresponds to gene frequencies of 0.817 and 0.183 for the type Ag(g)/Ag(c) alleles, respectively. After adjustment for age and body-mass index, it was found that the Ag(c) allele decreases the apo B level by 9.62 mg/dl and that the Ag(g) allele increases apo B by 0.43 mg/dl. Therefore, as much as 4.30% of the genetic variance for apo B level could be accounted for by the Ag(c,g) gene locus.

The increased plasma Lp(a): B lipoprotein particle concentration in angina pectoris is not associated with hypofibrinolysis.

The structural homology between plasminogen and apolipoprotein (a), the specific glycoprotein of Lp(a) lipoprotein, raises the possibility of a relationship between this lipoprotein and the plasma fibrinolytic system. The present study examines this proposal by studying 66 patients with angina pectoris. As compared to normal controls, the patients had raised concentrations of Lp(a): B lipoprotein particles. No correlation was found between circulating Lp(a): B and the fibrinolytic system. The pathogenic role of Lp(a): B lipoprotein seems therefore not mediated by its effect on the plasma fibrinolytic system.

A selective bi-site immunoenzymatic procedure for human Lp[a] lipoprotein quantification using monoclonal antibodies against apo[a] and apoB.

A selective bi-site ELISA assay procedure for quantification of Lp[a] lipoprotein in human plasma based on linkage of apo[a] to apoB is described. The lipoproteins referred to as apo[a]:B were captured by a mixture of two anti-apo[a] monoclonal antibodies (K07, K09) and were revealed by a mixture of six anti-apoB monoclonal antibodies coupled to peroxidase. Since apo[a] and plasminogen have striking similarities in protein structure, the selective binding of Lp[a]:B in our assay depended upon the marked difference in affinity of the K07 and K09 mixture for Lp[a]:B (Kd = 0.32 x 10(-10) M) versus plasminogen (Kd = 0.47 x 10(-7)M). The high sensitivity (the Lp[a]:B working range 0.06-0.40 micrograms/ml) and the use of anti-apoB as antibody tracer added to the selectivity of the assay. The expression of K07 and K09 epitopes determined by competitive inhibition method and the reactivity of Lp[a]:B particles measured by bi-site ELISA were similar on individual lipoproteins, independent to their plasma levels. The assay is precise, and intra- and interassay coefficients of variation were 4.7% and 9.6%, respectively. It yields quantitative Lp[a]:B values that correlate highly with Lp[a] levels obtained by electroimmunoassay with polyclonal antibody (r = 0.73) or with Lp[a] levels measured by the other bi-site ELISA using only K07 and K09 antibodies (r = 0.96). However, upon analyzing each individual plasma with an arbitrary Lp[a]-cut off of 15 mg/dl, evidence of the qualitative aspect of the lipoprotein was obtained. The group with Lp[a] less than 15 mg/dl had higher frequency of subjects (65%) with the ratio Lp[a]/Lp[a]:B above 1.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression, location and cross-reactivity of two antigenic sites on the amino terminal region of rabbit and human apolipoprotein A-I.

Rabbit apolipoprotein A-I (apo A-I) of molecular weight 27,612 contained 241 amino acids. In contrast to its human counterpart which has 3 methionine residues, the rabbit protein possesses only one and therefore produces 2 fragments after CNBr cleavage (CNBr I and II, NH2- and COOH-terminal, respectively). From a series of monoclonal antibodies raised against human apo A-I, 2 (A05 and A16) cross-reacted with rabbit apo A-I. In the present study, we show that A05 recognizes the rabbit CNBr I fragment while the integrity of the intermediate region between the 2 CNBr fragments (including the methionine residue) is required for the expression of the A16 antigenic determinant. Competition experiments were performed between human 125I-labelled high density lipoprotein (HDL) and a variety of preparations of human and rabbit apo A-I (including the purified and delipidated protein, complexes of dimyristoylphosphatidylcholine (DMPC) containing apo A-I, HDL subfractions and whole serum). The A05 antigenic determinant was expressed identically in all these fractions of both species. In contrast the A16 showed poor reactivity with delipidated apo A-I, the apparent affinity constant being about 100 times less than for HDL. These data suggest that phospholipids improve the recognition of apo A-I by the A16 antibody. The similar immunoreactivity of the human and rabbit proteins in the present study is consistent with the view that the NH2-terminal region contains the major portion activating lecithin:cholesterol acyltransferase.

Characterization of two monoclonal antibodies to human apolipoprotein A-I that also bind to rabbit apolipoprotein A-I. Application to ELISA evaluation of rabbit serum apo A-I.

Two monoclonal antibodies against human apolipoprotein A-I were characterized to recognize rabbit apo A-I. By immunoblotting we determined that while neither antibody reacted with the other rabbit apolipoproteins they both recognized all rabbit apo A-I isoforms. Cotitration revealed that each antibody bound to different apo A-I epitopes. Then we developed a noncompetitive enzyme-linked immunosorbent assay (ELISA) to measure the concentration of total apolipoprotein A-I in rabbit serum. The ELISA curves of the different lipoprotein class and serum showed the similarity of the expression of the apo A-I epitopes recognized in each of them and in serum. This assay, as opposed to other techniques, offers several advantages such as sensitivity, specificity, and simplicity and avoids the use of radioisotope.

Evidence for an interaction of lipoprotein lipase with artery wall proteoglycans.

1. Artery wall proteoglycans-lipoprotein lipase binding characteristics were studied using bovine milk 125I-labelled lipoprotein lipase (LPL) and chondroitin sulphate-dermatan sulphate proteoglycans (PGs) purified from pig aorta. 2. The binding process was studied either by a soluble assay (gel filtration) or by an immobilized proteoglycan assay (ELISA). 3. The binding process was reversible, saturable and occurred at a stoichiometry 1:1. 4. The binding process involved ionic interactions between the positively charged groups of LPL and the negatively charged groups of PG carbohydrate chains. 5. The complex PG-LPL may lead to the production of remnant lipoproteins and, thereby, contribute to cholesteryl ester accumulation in the arterial wall.

Electroimmuno- and immunonephelometric assays of apolipoprotein A-I by using a mixture of monoclonal antibodies.

The use of mixtures of well-defined monoclonal antibodies may represent a step forward in the standardization of immunochemical assays. We developed and optimized working conditions for using such a mixture to determine apolipoprotein A-I in human sera by two independent techniques (electroimmuno- and immunonephelometric-assays). Six monoclonal antibodies, each addressed to distinct epitopes located at the surface of apolipoprotein A-I, were used in combination to permit a reproducible measurement of the protein, without prior delipidation of samples. Parallel standard curves for a high-density lipoprotein subfraction (HDL3, the primary standard) and a reference serum (the secondary standard) were obtained. Within- and between-run coefficients of variation were acceptable for both methods. Apolipoprotein A-I concentrations, as measured in 60 subjects selected to present a large range of apolipoprotein content by electroimmunoassay (y1) and immunonephelometric assay (y2) with monoclonal antibodies, compared well with those measured by the same techniques but with polyclonal antibodies (x): r1 = 0.96, r2 = 0.99; y1 = 1.19x - 0.11 g/L, y2 = 0.98x. Comparison of results obtained by electroimmunoassay and immunonephelometric assay performed with monoclonal antibodies was also good: r = 0.96; y2 = 1.08y1 + 0.13 g/L.