Human apolipoprotein A-IV reduces secretion of proinflammatory cytokines and atherosclerotic effects of a chronic infection mimicked by lipopolysaccharide.

OBJECTIVE: Expression of human apolipoprotein (h-apo) A-IV in apoE-deficient (apoE(0)) mice (h-apoA-IV/E(0)) reduces susceptibility to atherosclerosis. Chronic infection mimicked by exposure to lipopolysaccharide (LPS) increases the size of atherosclerosis lesions in apoE(0) mice. Thus, we used h-apoA-IV/E(0) mice to determine whether h-apoA-IV plays a protective role after LPS administration. METHODS AND RESULTS: We injected apoE(0), h-apoA-IV/E(0), and C57Bl/6 (wild-type) mice intraperitoneally with either LPS or phosphate-buffered saline (PBS) every week for 10 weeks. Atherosclerotic lesions were significantly smaller in h-apoA-IV/E(0) mice treated with LPS than in their apoE(0) counterparts. The titers of IgG2a and IgG2b autoantibodies to oxidized low-density lipoprotein (LDL) were higher in the LPS-group of h-apoA-IV/E(0) mice than in apoE(0) mice, suggesting that the Th1 response is stronger in the presence of h-apoA-IV. Lymphocytes from the blood, liver, spleen, and thymus of h-apoA-IV/E(0) mice treated with LPS produced less IL-4, INF-gamma, and TNF-alpha proinflammatory cytokines than their apoE(0) counterparts. Furthermore, we demonstrated that recombinant h-apoA-IV blocks the LPS-induced stimulation of monocytes. CONCLUSIONS: The expression of h-apoA-IV in apoE(0) mice reduces the susceptibility to atherogenesis and decreases the secretion of proinflammatory cytokines after LPS administration.

Implication of natural killer T cells in atherosclerosis development during a LPS-induced chronic inflammation.

Atherosclerosis has many features of a chronic inflammatory disease. To evaluate the role of lipopolysaccharide (LPS), mimicking a systemic infection, we administered the endotoxin to apolipoprotein E (apoE)-deficient mice. LPS injections increase the atherosclerotic lesion size and the titer of plasma autoantibodies directed against oxidized low-density lipoprotein. We found that Th1 and Th2 T cells help the activation of B cells in the autoimmune response. The number of interleukin-4 producing natural killer T cells is highly increased in peripheral blood, liver, spleen and thymus cells, as well as in the atherosclerotic plaque of the LPS-treated mice. Finally, an important adventitial infiltrate of activated lymphocytes, sign of an advanced atherosclerosis, is observed only in the LPS-treated mice. Our results demonstrate that LPS administration aggravates atherosclerosis in apoE-deficient mice. LPS-injected apoE-deficient mice appear to be an excellent animal model to analyze the implementation of new therapeutic approaches in the treatment of atherosclerosis by manipulating immunological effectors.

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

Various studies have correlated apolipoprotein (apo) A-I, the major component high-density lipoprotein, with protection against development of cardiovascular disease. Although apoA-I expression has been previously detected in the liver and intestine, we have discovered that the human apoA-I gene is also expressed in the heart. Using transgenic (Tg) mice generated with the human apoA-I/C-III/A-IV gene cluster and Tg mice produced with just the 2.2 kb human apoA-I gene, we have detected significant levels of apoA-I expression in the heart. Furthermore, the detection of apoA-I expression in the hearts of human apoA-I Tg mice indicates that the minimal regulatory elements necessary for cardiac expression of the gene are located near its coding sequence. To determine if the apoA-I gene is also expressed in the human heart, similar analyses were performed, where apoA-I expression was found in both adult and fetal hearts. Furthermore in-depth investigation of the various regions of human and Tg mouse hearts revealed that the apoA-I mRNA was present in the ventricles and atria, but not in the aorta. In situ hybridization of Tg mouse hearts revealed that apoA-I expression was restricted to the cardiac myocyte cells. Finally, heart explants and cardiac primary culture experiments with Tg mice showed secretion of particles containing the human apoA-I protein, and metabolic labeling experiments have also detected a 28 kDa human apoA-I protein secreted from the heart. From these novel findings, new insights into the role and function of apoA-I can be extrapolated.

Human apoA-I/C-III/A-IV gene cluster transgenic rabbits: effects of a high-cholesterol diet.

We have generated transgenic rabbits that express the entire human apoA-I/C-III/A-IV gene cluster. As in humans, h-apoA-I and h-apoC-III were expressed in liver and intestine, whereas h-apoA-IV mRNA was detected in intestine only. Transgenic rabbits had significantly higher plasma total cholesterol, HDL-cholesterol and total phospholipid concentrations than non-transgenic littermates. In contrast to similar transgenic mice previously generated, which have gross hypertriglyceridemia, triglyceride concentrations were only moderately raised in transgenic rabbits. Plasma and HDL from transgenic rabbits were more effective than those from controls in promoting cholesterol efflux from cultured hepatoma cells. They had lower LCAT, lower CETP and higher PLTP activities than non-transgenic littermates. Cholesterol-feeding produced major increases in plasma lipids. The qualitative response to the diet was not modified by cluster expression. Human apoA-I concentration was halved by cholesterol-feeding, whereas h-apoC-III and h-apoA-IV concentrations were not significantly altered. Cholesterol efflux from hepatoma cells to plasma and HDL was not altered by the diet. Since lipoprotein metabolism of rabbits closely resembles that of humans, human apoA-I/C-III/A-IV transgenic rabbits may provide a reliable model for studies of the transcriptional regulation of the cluster, and for evaluating the effects of different agents on the expression of the three genes.

Regulation of the tissue-specific expression of transferrin gene.

Transferrin (Tf), the plasma protein involved in iron transport, seems to play complex physiological roles related to cell function, differentiation and proliferation. The protein is essentially synthesized in hepatocytes, but also in Sertoli cells, in the epithelial cells of the choroid plexus in rodents and in oligodendrocytes in all species analyzed. In this manuscript, we review the results obtained on Tf gene expression in the different cellular systems in which the protein is synthesized. In vitro and ex vivo experiments indicate that different combinations of transcription factors are necessary in different subsets of cells to achieve Tf tissue-specific expression. Several lines of transgenic mice were generated in which the expression of reporter genes is under the control of different Tf regulatory regions. More recently, transgenic mice were obtained using the complete human Tf gene and its 5' and 3' flanking sequences. These mice constitute the first model in which the physiological consequences of a specific Tf over expression in oligodendrocytes can be studied. Although much information is now available, further work is still necessary for a full understanding of the in vivo mechanisms responsible for the regulation of Tf gene expression.

Fructose intake increases hyperlipidemia and modifies apolipoprotein expression in apolipoprotein AI-CIII-AIV transgenic mice.

Fructose intake has increased steadily during the past two decades. The objective of this study was to determine the effect of fructose intake on lipid metabolism in apolipoprotein (apo) AI-CIII-AIV transgenic (Tg) mice that have severe hypertriglyceridemia and moderate hypercholesterolemia. Tg and control mice were fed for 9 mo a commercial nonpurified diet and had free access to water or 250 g/L fructose solution. In Tg mice, fructose intake increased triglycerides and cholesterol but did not induce insulin resistance. There were no differences in human hepatic apo AI and apo CIII mRNA levels in fructose-fed mice compared with untreated mice, but apo AIV mRNA was greater, indicating a differential expression of the apo AI and apo AIV genes in response to dietary perturbations. Interestingly, the plasma concentration of the three human apolipoproteins was enhanced in fructose-fed Tg mice compared with untreated Tg mice. Our data suggest that long-term fructose consumption had strong adverse effects in this hyperlipidemic mouse model.

Alternative splicing in the brain of mice and rats generates transferrin transcripts lacking, as in humans, the signal peptide sequence.

Transferrin (Tf), the iron-transport protein of vertebrate serum, is mainly synthesized in hepatocytes but is also found in other cell-types including oligodendrocytes. Our laboratory has characterized in a human oligodendrial cell line the presence of a new Tf transcript containing an alternative exon 1b replacing the classical exon 1 and conducting to the elimination of the signal peptide sequence. In this manuscript, we show by RT-PCR and 5'-RACE experiments that alternative transcripts also exist in mouse and rat and are found in brain mRNA preparations. Mouse alternative first exon is homologous to human exon 1b while rat Tf gene was found to use a new first exon named 1c. In all species, the alternative transcript does not contain the signal peptide sequence and possibly encode for a Tf protein devoid of signal peptide showing that this phenomenon is not restricted to human gene. We also present genomic sequence data from the previously unknown 5' genomic rat region, which allowed the alignment of the alternative exons 1 in the three species.

Expression, purification, and initial structural characterization of rat orphan nuclear receptor NOR-1 LBD domain.

NOR-1 is an orphan member of the nuclear receptor superfamily, which includes a group of transcription factors involved in the response to steroids, fatty acids, retinoic acids, and other lipophilic molecules. The NOR-1 subfamily (NR4), composed also of Nurr1 and Nurr77, has been implicated in cell proliferation, differentiation, apoptosis, chondrosarcomas, inflammation, and atherogenesis. The NOR-1 receptor is an orphan ligand receptor which acts over gene transactivation. No ligands, if such in fact exist, are known for this receptor. Recently, the three-dimensional structure of the homolog receptor Nurr1 has been solved using protein crystallography techniques. Surprisingly, the structure does not present either a typical cavity for ligand binding or a classical co-factor binding site in the ligand binding domain (LBD). To allow for structural studies of other members of NR4 subfamily, we have subcloned, overexpressed in Escherichia coli cells, purified, and characterized the rat orphan nuclear receptor NOR-1 LBD domain. We obtained NOR-1 LBD at a high degree of purity and with an overall yield of 3 mg/L of culture media. CD spectroscopic analysis shows a high alpha-helical secondary structure content (52%), similar to that of Nurr 1 LBD three-dimensional structure. Thermal denaturation monitored by UV absorption and CD spectroscopy suggests proper folding of recombinant NOR-1 LBD.

Myelination and motor coordination are increased in transferrin transgenic mice.

Myelin deficiency in the central nervous system (CNS) can cause severe disabling conditions. Most of the transgenic mice models overexpressing myelin components have limitations for investigators of myelin deficiency and myelin therapy as they severely alter CNS architecture. It has been postulated that transferrin (Tf) is involved in oligodendrocyte (OL) maturation and myelinogenesis. Because Tf is not an intrinsic myelin constituent, we decided to investigate if its overexpression could have an impact on the myelination process without affecting myelin integrity. We generated transgenic mice containing the complete human Tf gene specifically overexpressed in OLs. This overexpression leads to more than a 30% increase in myelin components, such as galactolipids, phospholipids, and proteins. Electron microscopy showed that myelin is structurally normal in terms of thickness and compaction. Behavior analysis showed that mice do not display significant modifications in their locomotion and cognitive and emotional abilities. Furthermore, in one of the genetic background, animals presented a significant increase in motor coordination. We did not find any modification in OL number during early postnatal development, suggesting that Tf does not act on OL proliferation. In addition, the levels of iron and ferritin remained unchanged in the brain of transgenic mice compared to control mice. Our findings indicate that, besides its known iron transport function, Tf is able to influence myelination process and induce behavioral improvements in mice.

Aspectos diagnósticos y funcionales de las Apolipoproteínas E y A-IV / Apolipoprotein E and A-IV diagnostic and functional aspects

En el presente trabajo se analizaron dos aspectos distintos del metabolismo lipoproteico. En el primero, en un estudio bioquímico clínico, se presentó un método rápido y simple de determinación de los polimorfismos de la apoE. La utilidad clínica de conocer el genotipo apoE fue evaluada correlacionando las isoformas de apoE con el espesor carotideo estimado con ecografía de ultrasonido. Los resultados mostraron que el genotipo apoE4 ejerce un efecto sobre la aterosclerosis carotídea, efecto que puede ser detectado antes que cualquier otro síntoma clínico. En la segunda parte de éste trabajo se ha analizado el metabolismo lipoproteico en ratones transgénicos expresando la apolipoproteína A-IV (apoA-IV) humana. Los resultados muestran que la apoA-IV tiene un rol pleiotrópico en el organismo, relacionado con la protección contra la aterosclerosis y con la regulación de la secreción gástrica

Aspectos diagnósticos y funcionales de las Apolipoproteínas E y A-IV / Apolipoprotein E and A-IV diagnostic and functional aspects

En el presente trabajo se analizaron dos aspectos distintos del metabolismo lipoproteico. En el primero, en un estudio bioquímico clínico, se presentó un método rápido y simple de determinación de los polimorfismos de la apoE. La utilidad clínica de conocer el genotipo apoE fue evaluada correlacionando las isoformas de apoE con el espesor carotideo estimado con ecografía de ultrasonido. Los resultados mostraron que el genotipo apoE4 ejerce un efecto sobre la aterosclerosis carotídea, efecto que puede ser detectado antes que cualquier otro síntoma clínico. En la segunda parte de éste trabajo se ha analizado el metabolismo lipoproteico en ratones transgénicos expresando la apolipoproteína A-IV (apoA-IV) humana. Los resultados muestran que la apoA-IV tiene un rol pleiotrópico en el organismo, relacionado con la protección contra la aterosclerosis y con la regulación de la secreción gástrica (AU)