Mario M. Zakin

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.

Antioxidative and antiatherosclerotic effects of human apolipoprotein A-IV in apolipoprotein E-deficient mice.

Abstract—Mice expressing human apolipoprotein A-IV (apoA-IV) mainly in the intestine were obtained in an apolipoprotein E-deficient (apoE0) background (apoA-IV/E0 mice). Quantification of aortic lesions and plasma lipid determination showed that compared with their control apoE0 counterparts, the apoA-IV/E0 mice are protected against atherosclerosis without an increase in HDL cholesterol. Because oxidized lipoproteins play an important role in atherogenesis, we tested whether the protection observed in these animals is accompanied by an in vivo reduction of the oxidation parameters. The lag time in the formation of conjugated dienes during copper-mediated oxidation, the aggregation state of LDL, and the presence of anti-oxidized LDL antibodies were measured. The presence of oxidized proteins in tissues and the presence of oxidation-specific epitopes in heart sections of atherosclerotic lesions were also analyzed. Except for lag time, the results showed that the oxidation parameters were reduced in the apoA-IV/E0 mice compared with the apoE0 mice. This suggests that human apoA-IV acts in vivo as an antioxidant. In addition, human apoA-IV accumulation was detected in the atherosclerotic lesions of apoA-IV/E0 mice, suggesting that apoA-IV may inhibit oxidative damage to local tissues, thus decreasing the progression of atherosclerosis.

Complete structure of the human transferrin gene. Comparison with analogous chicken gene and human pseudogene

The complete structure of the human transferrin gene is presented. This gene has a total size of about 33.5 kb and is organized in 17 exons separated by 16 introns. The chicken ovotransferrin gene has a size of 10.5 kb and is also organized in 17 exons and 16 introns. The analysis of the structure of the two genes confirm, at the gene level, that transferrins originated by a gene duplication phenomenon. Finally, the existence of a new member of the transferrin family, a human transferrin non-processed pseudogene is demonstrated.

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 (apoE0) mice (h-apoA-IV/E0) reduces susceptibility to atherosclerosis. Chronic infection mimicked by exposure to lipopolysaccharide (LPS) increases the size of atherosclerosis lesions in apoE0 mice. Thus, we used h-apoA-IV/E0 mice to determine whether h-apoA-IV plays a protective role after LPS administration. Methods and Results—We injected apoE0, h-apoA-IV/E0, 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/E0 mice treated with LPS than in their apoE0 counterparts. The titers of IgG2a and IgG2b autoantibodies to oxidized low-density lipoprotein (LDL) were higher in the LPS-group of h-apoA-IV/E0 mice than in apoE0 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/E0 mice treated with LPS produced less IL-4, INF-&ggr;, and TNF-&agr; proinflammatory cytokines than their apoE0 counterparts. Furthermore, we demonstrated that recombinant h-apoA-IV blocks the LPS-induced stimulation of monocytes. Conclusions—The expression of h-apoA-IV in apoE0 mice reduces the susceptibility to atherogenesis and decreases the secretion of proinflammatory cytokines after LPS administration.

Alternative splicing prevents transferrin secretion during differentiation of a human oligodendrocyte cell line.

Transferrin, the iron‐transport protein of vertebrate serum, is synthesized mainly in the liver, from which it is secreted into the blood. Transferrin is also synthesized in oligodendrocytes and is an early marker of their differentiation. We have analyzed the regulation of transferrin expression in HOG cells, a human oligodendrocyte cell line. Transferrin expression was correlated with the appearance of oligodendrocyte differentiation markers when cells were exposed to differentiation medium. In contrast to the protein expressed in hepatocytes or in Sertoli cells, transferrin was secreted by neither HOG cells nor immature rat primary oligodendrocytes in vitro. Moreover, transferrin appears to be localized in the cytosol and not in the secretory compartment, as is expected for secreted proteins. This transferrin localization was correlated with the synthesis of a specific transcript, resulting from an alternative splicing, which leads to the elimination of the signal peptide sequence. These results suggest the existence of a functional difference between transferrin synthesized in the brain and in other organs such as liver and testis. They are in accordance with the hypothesis that transferrin plays a specific role, other than iron transport, in oligodendrocyte maturation and in the myelination process. J. Neurosci. Res. 61:388–395, 2000.

Single intracranial injection of apotransferrin in young rats increases the expression of specific myelin protein mRNA

Transferrin (Tf) is a possible regulator of oligodendrocyte development in vitro (Espinosa de los Monteros et al., 1989). At least two different mechanisms may account for the effects of Tf on myelin synthesis. It may act as a trophic factor and enhance the formation of new myelin sheaths. Tf may also induce the synthesis of myelin proteins in the central nervous system. We recently demonstrated that a single intracranial injection of apotransferrin (aTf) in young rats induces an increased myelination (Escobar Cabrera et al., 1994). In the present study, we investigated the in vivo effect of aTf on the expression of mRNAs of specific myelin genes. Three‐day‐old rats were injected intracranially with aTf and killed at different ages after injection. Total brain RNA was isolated, and the expression of different mRNAs was analyzed by Northern blot. The amount of mRNAs of myelin basic protein and of 2′–3′ cyclic nucleotide 3′‐phosphohydrolase were markedly increased in the experimental animals, whereas myelin proteolipid protein mRNA did not show differences relative to controls. These results indicate that in the animals treated with aTf, there is a differential effect on the expression of certain specific myelin protein genes. They also suggest that aTf might exert its action at the posttranscriptional level and/or by direct transcriptional regulation of the genes. J. Neurosci. Res. 47:603–608, 1997.

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.

The apolipoprotein A-I/C-III/A-IV gene cluster: ApoC-III and ApoA-IV expression is regulated by two common enhancers

Genetic, epidemiological and clinical evidence have clearly demonstrated the importance of the human apolipoprotein (apo) A-I/C-III/A-IV gene cluster in lipid metabolism and heart attack. The transcriptional regulation of these genes determines the level of the encoded proteins and therefore influences the concentration of triglycerides and cholesterol. Here, we analyze the existence of transcription control elements in the 6.6 kb apoC-III/A-IV intergenic region and their influence on the expression of both genes. Two main positive common control elements were found to modulate apoC-III and apoA-IV expression in HepG2 and in Caco-2 cells: the previously described apoC-III enhancer, located 0.8 kb upstream from the cap site of the gene, and a newly detected activating region located in the center of the intergenic sequence. The activity of both elements is highly increased by the hepatic and intestinal transcription factor HNF-4. Analysis of a 641 bp fragment containing the central element showed that it has the properties of a tissue-specific enhancer. Liver nuclear proteins interact with seven DNA binding sites present in this enhancer and HNF-4 specifically interacts with one of these sites. A third positive element, situated immediately upstream from the apoA-IV minimal promoter, is also activated by HNF-4; however, this element is not involved in apoC-III expression. In addition, two negative regions were identified, one located near the apoA-IV gene and the other one between the apoC-III enhancer and the newly identified central enhancer. In conclusion, negative and positive control elements are located in the apoC-III/A-IV intergenic region, including two enhancers important for the expression of the two genes. These results add new evidence that common regulatory elements for the expression of the apoA-I, apoC-III and apoA-IV genes are interspersed throughout the cluster.

Expression of Human Apolipoprotein A-I/C-III/A-IV Gene Cluster in Mice Induces Hyperlipidemia but Reduces Atherogenesis

The apolipoprotein (apo)A-I/C-III/A-IV gene cluster is involved in lipid metabolism and atherosclerosis. Overexpression of apoC-III in mice causes hypertriglyceridemia and induces atherogenesis, whereas overexpression of apoA-I or apoA-IV increases cholesterol in plasma high density lipoprotein (HDL) and protects against atherosclerosis. Each gene has been studied alone in transgenic mice but not in combination as the entire cluster. To determine which phenotype is produced by the expression of the entire gene cluster, transgenic mice were generated with a 33-kb human DNA fragment. The results showed that the transgene contained the necessary elements to direct hepatic and intestinal expression of the 3 genes. In the pooled data, plasma concentrations were 257±9, 7.1±0.5, and 1.0±0.2 mg/dL for human apoA-I, apoC-III, and apoA-IV, respectively (mean±SEM). Concentrations of these apolipoproteins were higher in males than in females. Human apoA-I and apoC-III concentrations were positively correlated, suggesting that they are coregulated. Transgenic mice exhibited gross hypertriglyceridemia and accumulation of apoB48–containing triglyceride-rich lipoproteins. Plasma triglyceride and cholesterol concentrations were correlated positively with human apoC-III concentration, and HDL cholesterol was correlated with apoA-I concentration. In an apoE-deficient background, despite being markedly hypertriglyceridemic, cluster transgenic animals compared with nontransgenic animals showed a 61% reduction in atherosclerosis. This suggests that apoA-I and/or apoA-IV can protect against atherosclerosis even in the presence of severe hyperlipidemia. These mice provide a new model for studies of the regulation of the 3 human genes in combination.

Nucleotide sequence of a cDNA clone for human aldolase B

Two specific clones for human aldolase B were isolated from a human liver cDNA library using a rat aldolase B cDNA probe. The clones were identified by positive hybridization-selection and one of them was sequenced. The 127 C-terminal residues of the human protein were deduced from this nucleotide sequence analysis. They showed 92% homology with the corresponding previously published amino-acid sequence of rat liver aldolase B.