Immune-regulation of the apolipoprotein A-I/C-III/A-IV gene cluster in experimental inflammation

Apolipoprotein A-IV is a member of the apo A-I/C-III/A-IV gene cluster. In order to investigate its hypothetical coordinated regulation, an acute phase was induced in pigs by turpentine oil injection. The hepatic expression of the gene cluster as well as the plasma levels of apolipoproteins were monitored at different time periods. Furthermore, the involvement of the inflammatory mediators' interleukins 1 and 6 and tumor necrosis factor in the regulation of this gene cluster was tested in cultured pig hepatocytes, incubated with those mediators and apo A-I/C-III/A-IV gene cluster expression at the mRNA level was measured. In response to turpentine oil-induced inflammation, a decreased hepatic apo A-IV mRNA expression was observed (independent of apo A-I and apo C-III mRNA) not correlating with the plasma protein levels. The distribution of plasma apo A-IV experienced a shift from HDL to larger particles. In contrast, the changes in apo A-I and apo C-III mRNA were reflected in their corresponding plasma levels. Addition of cytokines to cultured pig hepatocytes also decreased apo A-IV and apo A-I mRNA levels. All these results show that the down-regulation of apolipoprotein A-I and A-IV messages in the liver may be mediated by interleukin 6 and TNF-alpha. The well-known HDL decrease found in many different acute-phase responses also appears in the pig due to the decreased expression of apolipoprotein A-I and the enlargement of the apolipoprotein A-IV-containing HDL.     

Gene and gene-product variation in the apolipoprotein A-I/C-III/A-IV cluster in the Dogrib Indians of the Northwest Territories.

The Dogrib, an Amerindian tribe residing in the Northwest Territories of Canada, were typed for DNA and protein polymorphism at the apolipoprotein A-I/C-III/A-IV gene cluster. Variation was seen at three previously described RFLPs detected with the enzymes SstI, PstI, and XmnI, though frequencies of these polymorphisms differ significantly from those reported in other populations. They exhibit no variation at two previously reported PvuII sites. No variation was seen in the APO A-I or APO A-IV gene products, with the Dogrib showing the most common isoelectric-focusing/immunoblot patterns of other world populations. Haplotype frequencies computed from inferred haplotypes and by maximum likelihood estimation did not differ significantly. The extent of nonrandom association of these sites is highly significant (P less than .00001), though pairwise analysis shows significance between the SstI and XmnI sites only. Levels of fasting triglyceride and fasting total cholesterol were determined for each individual. Analysis of covariance shows that fasting triglyceride levels in women vary significantly with the XmnI genotype. These results suggest that genetic variation at the APO A-I/C-III/A-IV gene cluster may be a useful tool for the study of quantitative lipoprotein variation in the Dogrib.     

Association between coronary heart disease and the apolipoprotein A-I/C-III/A-IV complex in a Japanese population

Several studies have reported that a variant allele (S₂) of the apolipoprotein (apo) A-I/C-III/A-IV complex is associated with hyperlipoproteinemia in some populations and that the frequency of this allele is two- to fivefold higher in patients with premature coronary heart disease (CHD) than in healthy controls. In the present study in a Japanese population, we were unable to confirm the association of the S₂ allele with either coronary heart disease or elevated serum apo C-III levels, as has been previously reported in Caucasians. No genotype difference was observed among the severity of coronary heart disease, as determined by the number of involved vessels (one, two and three vessel disease), compared to controls. In addition, the frequency of the S₂ allele among Japanese, in both CHD (0.328) and controls (0.369), was quite different from that in many other populations.     

Human apolipoprotein A-IV binds to apolipoprotein A-I/A-II receptor sites and promotes cholesterol efflux from adipose cells

Cholesterol efflux was studied in cultured mouse adipose cells after preloading with low density lipoprotein cholesterol. Exposure to complexes containing human apolipoprotein A-IV and L-alpha-dimyristoylphosphatidylcholine (DMPC) as well as to human lipoprotein particles containing apolipoprotein A-IV but not apolipoprotein A-I and particles containing apolipoproteins A-IV and A-I showed that both artificial and native apolipoprotein A-IV-containing particles were able to promote cholesterol efflux at 37 degrees C as a function of time and concentration. The half-maximal concentration was found to be 0.3 X 10(-6) M for apolipoprotein A-IV.DMPC complexes. Binding experiments performed in intact cells at 4 degrees C with labeled apolipoprotein A-IV.DMPC complexes showed the existence of specific binding sites, with a Kd value of 0.32 x 10(-6) M and a maximal binding capacity of 223,000 sites/cell. By cross-competition experiments with labeled and unlabeled complexes containing apolipoprotein A-IV, A-I, or A-II, it appeared that all three apolipoproteins bind to the same cell-surface recognition sites. It is suggested that apolipoprotein A-IV, which is present in the interstitial fluid surrounding adipose cells in vivo at concentrations similar to those required in vitro for the promotion of cholesterol efflux, plays a critical role in cholesterol removal from peripheral cells.     

Familial apolipoprotein A-I, C-III, and A-IV deficiency and premature atherosclerosis due to deletion of a gene complex on chromosome 11

The defect in a kindred with marked plasma high density lipoprotein (HDL) deficiency and premature atherosclerosis was examined. The homozygous proband died of coronary artery atherosclerosis at age 45 and had undetectable levels of plasma apolipoproteins A-I and C-III, proteins of HDL. In family studies 10 heterozygotes were identified whose mean apoA-I, apoC-III, apoA-IV, and HDL cholesterol levels were 67, 57, 65, and 62% of normal. These subjects were noted to have restriction fragment length polymorphisms following DNA digestion with a number of enzymes including BamHI, EcoRI, HindIII, XmnI, PstI, and PvuII, following hybridization with a probe spanning 1.1 kilobases approximately 2.5 kilobases 5' to the apoA-I gene. Cloning and sequence analysis of the abnormal allele indicated that the defect is due to the complete deletion of the apoA-I, -C-III, and -A-IV gene complex on chromosome 11, with both ends of the deletion being located in areas of highly repetitive DNA. The data support the concept of an independent role for HDL in the pathogenesis of atherosclerosis.     

Genetic polymorphism of human plasma apolipoprotein A-IV is due to nucleotide substitutions in the apolipoprotein A-IV gene

Genetic polymorphism of human plasma apolipoprotein A-IV has been detected by isoelectric focusing techniques followed by immunoblotting. The molecular basis for this apoA-IV polymorphism has been elucidated. Analysis of the protein coding sequences of the apoA-IV alleles 1 and 2 revealed a single G to T substitution in the apoA-IV-2 allele. The point mutation, occurring in a region highly conserved among the mouse, rat, and human A-IV apolipoproteins, converts the glutamine at position 360 of the mature protein to a histidine. This amino acid substitution adds one positive charge unit to the apoA-IV-1 isoprotein (pI 4.97) thus creating the more basic apoA-IV-2 isoprotein (pI 5.02). Computer analysis of the apoA-IV-2 allele revealed that the single G to T substitution results in the loss of a BbvI and a Fnu4HI restriction enzyme site and in the formation of a new restriction site for the enzyme SfaNI. Protein primary and secondary structure predictions were largely unaffected by this amino acid exchange. These results on the structure of the apoA-IV-1 and apoA-IV-2 alleles suggest that the three other rare isoproteins (apoA-IV-0, apoA-IV-3, and apoA-IV-4) are also due to nucleotide and subsequent amino acid substitutions in the apoA-IV sequence.     

Expression of human apolipoprotein A-I in Nicotiana tabacum

Several transgenic tobacco lines expressing human apolipoprotein A-I (ApoA-I) were obtained. Western blot analyses indicated the expression of the recombinant protein in plant organs at various stages of development, including senescent leaves. A cell line expressing human ApoA-I was established from a T₁ transgenic plant. Recombinant ApoA-I was isolated either from extracts of transgenic leaves and from the culture medium of transgenic cells using an antibody-based one-step procedure.     

Expression of human apolipoprotein A-I epitopes in high density lipoproteins and in serum

The expression and immunoreactivity of apolipoprotein (apo) A-I epitopes in high density lipoproteins (HDL) and serum has been investigated using two series of monoclonal antibodies (Mabs) which have been described elsewhere. Series 1 Mabs, identified as 3D4, 6B8, and 5G6, were obtained by immunization and screening with apoA-I, and series 2 Mabs, identified as 2F1, 4H1, 3G10, 4F7, and 5F6, were obtained by immunization and screening with HDL. These Mabs were characterized with respect to their binding to HDL particles in solution. In series 2 Mabs, 2F1, 3G10, and 4F7, which react with apoA-I CNBr-fragments 1 and 2, could precipitate 100% of 125I-labeled HDL, while 4H1 and 5F6, which react with CNBr fragments 1 and 3, precipitated 90 and 60% of 125I-labeled HDL, respectively. Therefore, three distinct epitopes mapped to CNBr fragments 1 and 2 have been identified which are expressed on all HDL particles, indicating that several antigenic do mains exist on apoA-I which have the same conformation on all apoA-I-containing lipoproteins. The Mabs reacting at these sites have significantly higher affinity constants for 125I-labeled HDL than those that failed to precipitate 100% of HDL. This suggests that the high affinity Mabs react with apoA-I epitopes that are both expressed on all lipoproteins and located in thermo-dynamically stable regions of the molecules. All Mabs from series 1 precipitated 35% or less of 125I-labeled HDL prepared from freshly collected serum, but the proportion of HDL particles expressing the epitopes for these Mabs doubled or more upon serum storage at 4 degrees C. The time course of the alteration of apoA-I antigen in vitro was measured in three normolipemic donors. Upon storage of serum at 4 degrees C, the immunoreactivity of series 2 Mabs (4H1, 3G10) remained unchanged. However, the immunoreactivity of series 1 Mab 3D4 increased linearly at 38%/day for 4 weeks and by 12 weeks had plateaued at about 280-fold compared to day 1. The immunoreactivity of other series 1 Mabs also increased significantly with time in vitro. This process was partially inhibited in the presence of EDTA and by addition of antioxidants, however, the exact molecular nature of this in vitro alteration of apoA-I antigen was not identified.     

Helper-dependent adenoviral vector-mediated long-term expression of human apolipoprotein A-I reduces atherosclerosis in apo E-deficient mice

Apolipoprotein A-I (APOA-I) is the major protein component of high-density lipoproteins (HDL). It has been shown that over-expression of human APOA-I increases HDL cholesterol and decreases atherosclerosis. We constructed a helper-dependent adenoviral (HD-Ad) vector that contains the entire human APOA-I gene (hgAI). Intravenous delivery of 1x10(13) viral particles/kg of this vector was followed by high levels of human APOA-I expression (up to 200 mg/dl) in the absence of detectable hepatic toxicity. We treated apo E-deficient mice with the hgAI vector and fed them either with a high-fat diet or with regular chow. As a control, two groups of mice were treated with PBS. The apo E-deficient mice treated with the hgAI vector showed supraphysiological levels of expression of human APOA-I at week 4 and high levels of HDL cholesterol compared to the control groups. Analysis of aortic atherosclerotic lesions 20 weeks after treatment, showed a significant reduction of lesion size in the treated mice with both diets. In conclusion, liver-directed gene transfer of human APOA-I using a HD-Ad vector resulted in a reduction of the development of atherosclerosis with the absence of significant toxicity.     

Methylation patterns of the human apoA-I/C-III/A-IV gene cluster in adult and embryonic tissues suggest dynamic changes in methylation during development.

We describe here a detailed analysis of the methylation patterns of the apoC-III and apoA-IV genes in adult and embryonic tissues. Together with previously reported data on the human apoA-I gene (4), the results presented here constitute a comprehensive study on the methylation pattern of the apoA-I/C-III/A-IV gene cluster. The two genes (apoC-III and apoA-IV) display tissue-specific methylation patterns that correlate with their activity. This gene-specific methylation pattern indicates that the apoA-I/C-III/A-IV gene cluster is not one entity with respect to methylation. The cluster is almost entirely methylated in tissues that do not express any of the genes; however, individual gene regions are unmethylated in the tissue of expression. A comparison of the observed methylation patterns in adult tissues with those in embryonic tissues suggests that the mature tissue-specific methylation patterns are a result of an interplay between demethylation and de novo methylation events in the embryo. These changes in DNA methylation include demethylation in the early embryo followed by de novo methylation at later stages. A second round of tissue-specific demethylation and methylation de novo occurs in the late embryo as well. Evidence presented here supports the idea that CpG islands are protected in general from methylation de novo by a built-in signal and not by CpG density per se.     

Obesity induced by a high-fat diet downregulates apolipoprotein A-IV gene expression in rat hypothalamus

Apolipoprotein A-IV (apo A-IV) is an anorectic protein produced in the intestine and brain that has been proposed as a satiety signal. To determine whether diet-induced obesity alters apo A-IV gene expression in the intestine and hypothalamus, rats were fed a high-fat (HF), low-fat (LF), or standard chow (CHOW) diet for 2, 4, 6, 8, or 10 wk. Rats fed the HF diet had significantly greater body weights than rats given the LF and CHOW diets. Intestinal and plasma apo A-IV levels were comparable across dietary groups and time. LF and CHOW rats had comparable hypothalamic apo A-IV mRNA across the course of the experiment. However, HF rats had a slow and progressive diminution in hypothalamic apo A-IV mRNA over time that became significantly lower than that of LF or CHOW rats by 10 wk. Intragastric infusion of lipid emulsion to animals that were fasted overnight significantly stimulated hypothalamic apo A-IV mRNA in LF and CHOW rats but had no effect in HF rats. These results demonstrate that chronic consumption of a HF diet significantly reduces apo A-IV mRNA levels and the response of apo A-IV gene expression to dietary lipids in the hypothalamus. This raises the possibility that dysregulation of hypothalamic apo A-IV could contribute to diet-induced obesity.     

Prebeta high density lipoprotein has two metabolic fates in human apolipoprotein A-I transgenic mice

We compared the in vivo metabolism of prebeta HDL particles isolated by anti-human apolipoprotein A-I (apoA-I) immunoaffinity chromatography (LpA-I) in human apoA-I transgenic (hA-I Tg) mice with that of lipid-free apoA-I (LFA-I) and small LpA-I. After injection, prebeta LpA-I were removed from plasma more rapidly than were LFA-I and small LpA-I. Prebeta LpA-I and LFA-I were preferentially degraded by kidney compared with liver; small LpA-I were preferentially degraded by the liver. Five minutes after tracer injection, 99% of LFA-I in plasma was found to be associated with medium-sized (8.6 nm) HDL, whereas only 37% of prebeta tracer remodeled to medium-sized HDL. Injection of prebeta LpA-I doses into C57Bl/6 recipients resulted in a slower plasma decay compared with hA-I Tg recipients and a greater proportion (>60%) of the prebeta radiolabel that was associated with medium-sized HDL. Prebeta LpA-I contained one to four molecules of phosphatidylcholine per molecule of apoA-I, whereas LFA-I contained less than one. We conclude that prebeta LpA-I has two metabolic fates in vivo, rapid removal from plasma and catabolism by kidney or remodeling to medium-sized HDL, which we hypothesize is determined by the amount of lipid associated with the prebeta particle and the particle's ability to bind to medium-sized HDL.     

Expression and secretion of human apolipoprotein A-I in the heart

Nuclear envelope-peripheral heterochromatin fractions contain multiple histone kinase activities. In vitro assays and amino-terminal sequencing show that one of these activities co-isolates with heterochromatin protein 1 (HP1) and phosphorylates histone H3 at threonine 3. Antibodies recognizing this post-translational modification reveal that in vivo phosphorylation at threonine 3 commences at early prophase in the vicinity of the nuclear envelope, spreads to pericentromeric chromatin during prometaphase and is fully reversed by late anaphase. This spatio-temporal pattern is distinct from H3 phosphorylation at serine 10, which also occurs during cell division, suggesting segregation of differentially phosphorylated chromatin to different regions of mitotic chromosomes.     

Effect of apolipoprotein activators on the specificity of lecithin:cholesterol acyltransferase: determination of cholesteryl esters formed in A-I/C-III deficiency.

Although it is known that plasma lecithin:cholesterol acyltransferase (LCAT) is activated by several apolipoproteins (apo) including A-I, C-I, D, A-IV, and E, it is not clear what the physiological importance of having different apolipoprotein activators is. One possible explanation is that the activation by different apolipoproteins may result in the utilization of different species of phosphatidylcholine (PC), leading to the formation of different species of cholesteryl esters (CE). In order to determine this possibility, we analyzed the molecular species composition of PC and CE in two patients with familial deficiency of apoA-I and apoC-III. The LCAT activity, assayed by three different procedures, was found to be 36-63% of the control value. The lower LCAT activity, however, was due to deficiency of the enzyme rather than the absence of apoA-I. The patients' plasma was relatively enriched with sn-2 18:2 PC species reflecting the partial deficiency of LCAT activity. The fatty acid composition of plasma CE was not significantly different from that of controls. HPLC analysis of labeled CE formed after incubation of plasma with [C14]cholesterol showed no significant difference in the species of CE synthesized by the LCAT reaction. The transfer of pre-existing as well as newly formed CE from HDL to the apoB-containing lipoproteins was accelerated compared to control plasma. These results show that the absence of apoA-I does not significantly affect either the activity or the specificity of LCAT, and that the other apolipoprotein activators can substitute adequately for it.     

A method to screen apolipoprotein polymorphisms in whole plasma: description of apolipoprotein A-IV variants in dyslipidemias and a reassessment of apolipoprotein A-I in Tangier disease

A sensitive and rapid immunological detection method was used to screen for apolipoprotein A-IV variants. Antibodies to human lymph chylomicron or plasma apolipoprotein A-IV, and plasma apolipoprotein A-I were raised in rabbits. Antibodies to apolipoprotein A-I or apolipoprotein A-IV were shown to be monospecific to their respective antigens by reactivity against human chylomicron apolipoproteins by immunoblot analysis. Plasma samples were obtained from dyslipidemic subjects from the Lipid Research Clinic of Columbia University. The plasma samples were isoelectrically focused (pH 4-6) on slab gels. Plasma proteins were then transferred to nitrocellulose paper for immunoblotting. Apolipoprotein A-IV polymorphism was determined by specific immunological detection of apolipoprotein A-IV. Identical apolipoprotein A-IV isoprotein patterns were observed when either antibodies to lymph or plasma apolipoprotein A-IV were used for immunoblotting. All the dyslipidemic plasma samples screened contained the two major and one or two minor isoproteins of normal plasma. In two instances, new apolipoprotein A-IV variants having an additional isoform were detected. One subject was hypertriglyceridemic (triacylglycerols = 342 mg/dl, cholesterol = 251 mg/dl) and had an additional major acidic apolipoprotein A-IV isoform. Another subject with mild hypocholesterolemia (triacylglycerols = 209 mg/dl, cholesterol = 120 mg/dl) was found to have additional major and minor basic apolipoprotein A-IV isoforms. The specificity of this technique allows detection of polymorphism of apolipoproteins of similar isoelectric points by use of a single dimension isoelectric focusing gel. This technique also demonstrated the presence of altered apolipoprotein A-I isoforms in the plasma of a patient with Tangier disease. These isoforms were previously identified as isoforms 2 and 4 of normal plasma by use of two-dimensional gel electrophoresis. However, by use of this new technique and careful evaluation of previously published two-dimensional gels, we now identify these apolipoprotein A-I isoforms as being more acidic than those of normal plasma.     

DNA polymorphisms of the apolipoprotein B and A-I/C-III genes are associated with variations of serum low density lipoprotein cholesterol level in childhood.

A number of restriction fragment length polymorphisms (RFLPs) of the apolipoprotein B (apoB) and apolipoprotein A-I/C-III(apoA-I/C-III) genes have been found to be associated with serum lipoprotein levels in many adult populations. In order to examine whether these genetic polymorphisms influence serum lipoprotein levels in childhood and adolescence, we determined the apoB XbaI and apoA-I/C-III SstI genotypes and serum lipoprotein concentrations in 307 healthy Finns aged 9 to 21 years. In the age groups of 9, 12, and 15 years, subjects homozygous for the X2 allele (the XbaI site present) of the apoB gene had mean serum low-density lipoprotein (LDL) cholesterol levels (3.69, 3.43, and 3.15 mmol/l, respectively) that were 12-20% higher than those in subjects homozygous for the absence of this allele (3.08, 3.02, and 2.80 mmol/l, respectively). This association was more significant in males than in females. At the age of 9 to 18 years, subjects carrying the S2 allele (SstI site present) of the apoA-I/C-III gene complex had an approximately 6-15% higher mean serum LDL-cholesterol level than those homozygous for its absence. The combined genotype X2+S2+ (the simultaneous presence of the X2 allele and the S2 allele) was associated with an even more marked elevation of serum LDL-cholesterol level than either allele alone. As an example, the serum LDL cholesterol concentration was 20% higher in 9-year-old subjects with at least one X2 and one S2 allele than in those without either allele (3.55 vs. 2.97 mmol/l, P less than 0.005). The S2 allele was found to be significantly more frequent in eastern than in western Finland, whereas no significant areal differences were seen in the occurrence of the X2 allele. In conclusion, genetic variations of the apoB and apoA-I/C-III gene loci influence serum lipoprotein concentrations already in childhood.     

Secretion of apolipoprotein A-I in lipoprotein particles following transfection of the human apolipoprotein A-I gene into 3T3 cells

Apolipoprotein A-I (apoA-I) is the major protein constituent of plasma high density lipoproteins (HDL). To examine apoA-I processing and secretion, the human apoA-I gene (2.2-kilobase PstI-PstI fragment) linked to the mouse metallothionein promoter was transfected by electroporation into NIH 3T3 fibroblasts along with the plasmid pSV2 neo, which confers neomycin resistance. Transfected cells were selected for neomycin resistance and screened for the ability to produce apoA-I by enzyme-linked immunosorbent assay. In the absence of lipids in the medium, selected 3T3 cells secreted apoA-I, mainly in the proprotein form, at density greater than 1.25 g/ml. Following incubation of cells with lipids, and subsequent washing with lipid-free medium, apoA-I was recovered in the HDL region (1.063-1.21 g/ml) as well as in the 1.21 g/ml infranatant. Examination of the HDL fraction by electron microscopy revealed round particles, 10-21 nm in diameter. These data indicate that human apoA-I secreted by transfected 3T3 fibroblasts can assemble into lipoprotein particles under the appropriate conditions.