Nadine Baroukh

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.

Analysis of Apolipoprotein A5, C3, and Plasma Triglyceride Concentrations in Genetically Engineered Mice

Objective—Both the apolipoprotein A5 and C3 genes have repeatedly been shown to play an important role in determining plasma triglyceride concentrations in humans and mice. In mice, transgenic and knockout experiments indicate that plasma triglyceride levels are strongly altered by changes in the expression of either of these 2 genes. In humans, common polymorphisms in both genes have also been associated with plasma triglyceride concentrations. These similar findings raised the issue of the relationship between these 2 genes and altered triglycerides. Methods and Results—To address this issue, we generated independent lines of mice that either overexpressed (“double transgenic”) or completely lacked (“double knockout”) both apolipoprotein genes. We report that both “double transgenic” and “double knockout” mice display normal triglyceride concentrations compared with overexpression or deletion of either gene alone. Furthermore, we find that human ApoAV plasma protein levels in the “double transgenic” mice are ≈500-fold lower than human ApoCIII levels, supporting ApoAV as a potent triglyceride modulator despite its low concentration. Conclusions—Together, these data support that APOA5 and APOC3 independently influence plasma triglyceride concentrations but in an opposing manner.

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.

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.

Expression of human apolipoprotein A-I/C-III/A-IV gene cluster in mice reduces atherogenesis in response to a high fat-High cholesterol diet

We have previously generated transgenic (Tg) mice expressing the human apolipoprotein (apo) A‐I/C‐III/A‐IV gene cluster. This expression induced hyperlipidemia but reduced atherosclerotic lesions in genetically modified mice lacking apoE. Atherosclerosis is a multifactorial process and environmental factors such as diet play significant roles in its development. We examined here how an atherogenic diet influences the expression of the human genes and the characteristics of the Tg mice. Our results indicate that a high fat‐high cholesterol diet up‐regulates the intestinal expression of the three genes and the concentration of the three proteins in plasma. Cholesterol concentration was highly increased in the non‐high density lipoprotein (HDL) fraction, and less, although significantly, in the HDL fraction. Tgs showed a 65% reduction in diet‐induced aortic lesions compared with non‐Tg mice. Atherogenic diet increases the expression of the genes encoding the scavenger receptor class B type I (SR‐BI) and ATP binding cassette transporter 1 (ABCA1) proteins. As cholesterol efflux mediated by SR‐BI or by ABCA1 was enhanced in Tg mice fed an atherogenic diet, we can hypothesize that increased reverse cholesterol transport is the basis of the protective mechanism observed in these animals. In conclusion, we present evidence that the expression of the human gene cluster in mice protects against atherogenesis in response to an atherogenic diet.

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.

Human apolipoprotein A-IV reduces gastric acid secretion and diminishes ulcer formation in transgenic mice.

We have investigated the involvement of human apolipoprotein A‐IV (apoA‐IV) in gastric acid secretion and ulcer formation in recently generated apoA‐IV transgenic mice. Compared to control littermates, transgenic animals showed a gastric acid secretion decreased by 43–77% whereas only slight variations were observed in the different cell population densities within the gastric mucosa. In addition, no variation in gastrin levels was observed. Transgenics were protected against indomethacin‐induced ulcer formation, with lesions diminishing by 45 to 64% compared to controls. These results indicate that endogenous apoA‐IV expression can regulate gastric acid secretion and ulcer development.