Opposite regulation of human versus mouse apolipoprotein A-I by fibrates in human apolipoprotein A-I transgenic mice.

The regulation of liver apolipoprotein (apo) A-I gene expression by fibrates was studied in human apo A-I transgenic mice containing a human genomic DNA fragment driving apo A-I expression in liver. Treatment with fenofibrate (0.5% wt/wt) for 7 d increased plasma human apo A-I levels up to 750% and HDL-cholesterol levels up to 200% with a shift to larger particles. The increase in human apo A-I plasma levels was time and dose dependent and was already evident after 3 d at the highest dose (0.5% wt/wt) of fenofibrate. In contrast, plasma mouse apo A-I concentration was decreased after fenofibrate in nontransgenic mice. The increase in plasma human apo A-I levels after fenofibrate treatment was associated with a 97% increase in hepatic human apo A-I mRNA, whereas mouse apo A-I mRNA levels decreased to 51%. In nontransgenic mice, a similar down-regulation of hepatic apo A-I mRNA levels was observed. Nuclear run-on experiments demonstrated that the increase in human apo A-I and the decrease in mouse apo A-I gene expression after fenofibrate occurred at the transcriptional level. Since part of the effects of fibrates are mediated through the nuclear receptor PPAR (peroxisome proliferator-activated receptor), the expression of the acyl CoA oxidase (ACO) gene was measured as a control of PPAR activation. Both in transgenic and nontransgenic mice, fenofibrate induced ACO mRNA levels up to sixfold. When transgenic mice were treated with gemfibrozil (0.5% wt/wt) plasma human apo A-I and HDL-cholesterol levels increased 32 and 73%, respectively, above control levels. The weaker effect of this compound on human apo A-I and HDL-cholesterol levels correlated with a less pronounced impact on ACO mRNA levels (a threefold increase) suggesting that the level of induction of human apo A-I gene is related to the PPAR activating potency of the fibrate used. Treatment of human primary hepatocytes with fenofibric acid (500 microM) provoked an 83 and 50% increase in apo A-I secretion and mRNA levels, respectively, supporting that a direct action of fibrates on liver human apo A-I production leads to the observed increase in plasma apo A4 and HDL-cholesterol.

Negative regulation of Apo A-I gene expression by retinoic acid in rat hepatocytes maintained in a coculture system.

Rat hepatocytes cocultured with rat liver epithelial cells (RLEC) were used to investigate the influence of all-trans retinoic acid (RA) on the regulation of apolipoproteins (Apo) A-I and A-II gene expression, the major protein constituent of high-density lipoproteins. In contrast to rat hepatocytes in conventional primary culture, Apo A-I and Apo A-II gene expression remained high and stable for several days in parenchymal cells in coculture. Treatment of cocultured rat hepatocytes with RA resulted in a specific decrease in Apo A-I mRNA levels whereas no marked difference in Apo A-II mRNA levels was observed. Such a negative effect of RA was already detected as early as 2 days of treatment and was effective for the entire experimental period (6 days). As controls, RARbeta mRNA levels increased whereas those of GAPDH mRNA were not affected by the RA treatment. The decrease in Apo A-I mRNA levels was associated with lower amounts of Apo A-I secreted in the culture medium within day 1 of treatment. This effect required active transcription and protein synthesis. These results show that, contrary to primary pure hepatocyte cultures and hepatoma cell lines, cocultures of rat hepatocytes reproduce the in vivo results suggesting that only well differentiated hepatocytes may correctly respond to RA. Furthermore, they demonstrate that RA can directly act on hepatocytes and differently affect Apo A-I and Apo A-II gene expression.

The phosphoinositide pathway of lymphoid cells: labeling after permeabilization by alveolysin, a bacterial sulfhydryl-activated cytolysin.

An improved method allowing incorporation of [3H]myo-inositol into the phosphoinositide pool of human lymphoid cells is described. The procedure devised involves cell permeabilization with a thiol-activated membranolytic toxin, alveolysin, and optimization of the phosphoinositide labeling and extraction. In these conditions 4 to 10% of the added [3H]myo-inositol is found intracellularly and half of this amount (2-5%) is incorporated into the phosphoinositide pool in only 1 h as compared to the classical 0.2 to 0.3% incorporation obtained after 10 to 20 h. The integrity of coupling between receptors and phospholipase C was assessed by the inositol phosphate production after cell stimulation by various agonists.

A 700-bp fragment of the human antithrombin III promoter is sufficient to confer high, tissue-specific expression on human apolipoprotein A-II in transgenic mice.

The human antithrombin III-encoding gene (hAT-III) promoter (phAT-III) was used to generate transgenic mice producing a human hepatic apolipoprotein, apolipoprotein A-II (hApoA-II). Integration of the transgene into the mouse genome resulted in the efficient production of hApoA-II in plasma, reaching up to 0.40 g/l in two transgenic lines. The human ApoA-II mRNA was detected at high levels, both in the liver and in the kidney of transgenic mice. The rat AT-III gene shows the same expression pattern. In contrast, as previously described, the same promoter permitted the expression of the SV40 large T antigen only in the liver of transgenic mice. In view of the extra-hepatic distribution of the ApoA-II mRNA, a preliminary characterization of the hAT-III proximal promoter (phAT-III), driving the expression of the transgene, was realized. Using DNase I footprinting analysis with liver nuclear extracts, four protected regions (I-IV) were identified in the first 175 bp of the 5' region of hAT-III. Electrophoretic mobility shift assays performed with liver and kidney nuclear extracts indicate that region III (nucleotides (nt) -67 to -90) interacts with the liver-enriched HNF4 nuclear factor. Furthermore, our data suggest that region I (nt -123 to -138) interacts with the liver-enriched HNF3 transcription factor family, both in liver and kidney. Taken together, these results demonstrate that phAT-III is a useful tool to create transgenic mice producing high plasma levels of a human apolipoprotein due to expression of the transgene in liver and kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiogenesis and gene therapy in man: dream or reality?

Preclinical studies indicate that angiogenic growth factors can stimulate the development of collateral arteries in animal models of peripheral or myocardial ischaemia, a concept termed 'therapeutic angiogenesis'. The goal of this review is to provide a brief overview of the advantages and disadvantages of gene versus recombinant protein therapy for therapeutic angiogenesis. We also discuss different options for delivering genes to patients, including plasmids and modified viral vectors. Recently, the safety and potential utility of gene therapy for ischaemic disease were demonstrated in 3 clinical trials involving the delivery of plasmid DNA encoding the 165 amino acid isoform of human vascular endothelial growth factor (phVEGF165), a factor that specifically promotes the proliferation and migration of vascular endothelial cells. Two trials involved the administration of phVEGF165 for peripheral arterial disease. In one trial, the plasmid was administered to the arterial wall from a hydrogel-coated angioplasty balloon, while a second trial examined the direct injection of phVEGF165 into the skeletal muscle of the affected limb. More recently, phVEGF165 was directly injected into ischaemic myocardium. In all these trials, it appears that administration of phVEGF165 led to improvements in tissue perfusion.

Angiogenesis and gene therapy in man: dream or reality?

Preclinical studies indicate that angiogenic growth factors can stimulate the development of collateral arteries in animal models of peripheral or myocardial ischaemia, a concept termed 'therapeutic angiogenesis'. The goal of this review is to provide a brief overview of the advantages and disadvantages of gene versus recombinant protein therapy for therapeutic angiogenesis. We also discuss different options for delivering genes to patients, including plasmids and modified viral vectors. Recently, the safety and potential utility of gene therapy for ischaemic disease were demonstrated in 3 clinical trials involving the delivery of plasmid DNA encoding the 165 amino acid isoform of human vascular endothelial growth factor (phVEGF165), a factor that specifically promotes the proliferation and migration of vascular endothelial cells. Two trials involved the administration of phVEGF165 for peripheral arterial disease. In one trial, the plasmid was administered to the arterial wall from a hydrogel-coated angioplasty balloon, while a second trial examined the direct injection of phVEGF165 into the skeletal muscle of the affected limb. More recently, phVEGF165 was directly injected into ischaemic myocardium. In all these trials, it appears that administration of phVEGF165 led to improvements in tissue perfusion.

Partial restoration of all trans retinoic acid (ATRA) sensitivity by compactin in ATRA-resistant leukemic cells (ATRA-R HL-60).

The resistance to all trans retinoic acid (ATRA) differentiating treatment is a consequence, in most of the cases, of either increased catabolism or down regulation of ATRA uptake. Recently, we have shown that ATRA efficiency to differentiate HL-60 cells was enhanced about 30 times after its incorporation into Low Density Lipoprotein (ATRA-LDL). Here, we attempted to differentiate the ATRA-resistant HL-60 cells by ATRA-LDL at high concentrations up to 10 microM. No significant differentiating effect was observed, although the LDL receptor sites were evidenced in these cells. To increase the number of LDL receptors, the cells were pre-incubated in lipoprotein-deprived serum medium and compactin (2 microM), both ATRA and ATRA-LDL induced gradual increase of cell differentiation (35%+/-1 and 51.5%+/-5 at 10 microM of ATRA and ATRA-LDL respectively). At 2 and 8 microM, the intracellular concentrations of ATRA were respectively three and four times higher when incorporated into LDL. In addition, ATRA-LDL, in the medium, was better protected against degradation than ATRA. The surprising restoration of free ATRA sensitivity after treatment with compactin suggested the implication of new mechanisms unrelated to the LDL-receptor endocytosis but involving the non-sterol pathway.

Opposite in vitro and in vivo regulation of hepatic apolipoprotein A-I gene expression by retinoic acid. Absence of effects on apolipoprotein A-II gene expression.

We studied the pharmacological potential of retinoids to modulate apolipoprotein (apo) A-I and apoA-II gene expression and production in vitro in the human cell line HepG2 as well as in primary cultures of adult rat hepatocytes and in vivo in the rat. In HepG2 cells, addition of all-trans retinoic acid (RA) doubled apoA-I mRNA within 24 hours and protein secreted in the culture medium after 48 hours. The induction of apoA-I mRNA by RA was completely blocked by actinomycin D, suggesting that RA acts at the transcriptional level in HepG2 cells. In primary cultures of rat hepatocytes, addition of RA increased apoA-I mRNA in a dose- and time-dependent manner as well as the secretion of apoA-I protein. Similar changes in apoA-I mRNA were observed with 9-cis RA. However, in vivo, hepatic apoA-I mRNA levels decreased after a single administration of RA at 10 mg/kg and remained low after prolonged treatment or at a higher dose, and serum apoA-I concentrations did not change. Furthermore, RA treatment did not substantially affect apoA-II mRNA levels or protein secretion either in vitro or in vivo. As a control, RA receptor-beta mRNA levels increased after RA both in vitro and in vivo. In conclusion, RA treatment selectively induces apoA-I and not apoA-II expression in vitro but not in vivo. These results therefore show additional regulatory effects of RA on apoA-I gene expression in vivo and raise questions about the usefulness of RA in the treatment of atherosclerosis.

Regulation of rat liver apolipoprotein A-I, apolipoprotein A-II and acyl-coenzyme A oxidase gene expression by fibrates and dietary fatty acids.

The regulation by fibrates and dietary fatty acids of the hepatic gene expression of apolipoproteins (apo) A-I and A-II, the major protein constituents of high-density lipoproteins, as well as of acyl-CoA oxidase, the rate-limiting enzyme of the peroxisomal beta-oxidation pathway, was studied in vivo in the rat and in vitro in primary cultures of rat hepatocytes. In primary hepatocytes, different fibrates decreased apo A-I and increased acyl-CoA oxidase mRNA levels, whereas apo A-II mRNA only decreased in level after treatment with fenofibric acid, but not after bezafibrate, gemfibrozil or Wy-14643 treatment. Treatment with fenofibric acid counteracted the increase in apo A-I mRNA levels observed after dexamethasone or all-trans retinoic acid treatment, whereas simultaneous addition of fenofibric acid together with all-trans retinoic acid or dexamethasone resulted in a superinduction of acyl-CoA oxidase mRNA. Addition of the n-3 polyunsaturated fatty acids (PUFAs), docosanohexaenoic acid and eicosanopentaenoic acid, or the fatty acid derivative alpha-bromopalmitate, decreased apo A-I and increased acyl-CoA oxidase mRNA in a dose-dependent and time-dependent manner, whereas apo A-II mRNA did not change significantly. Nuclear run-on experiments demonstrated that fenofibric acid and alpha-bromopalmitate decreased apo A-I and increased acyl-CoA oxidase gene expression at the transcriptional level. When rats were fed isocaloric diets enriched in saturated fat (hydrogenated coconut oil), n-6 PUFAs (safflower oil) or n-3 PUFAs (fish oil), a significant decrease in liver apo A-I and apo A-II mRNA levels was only observed after fish oil feeding. Compared to feeding low fat, liver acyl-CoA oxidase mRNA increased after fat feeding, but this effect was most pronounced (twofold) in rats fed fish oil. Results from these studies indicate that fish oil feeding reduces rat liver apo A-I and apo A-II gene expression, similar to results obtained after feeding fenofibrate. Fibrates and n-3 fatty acids (and the fatty acid derivative, alpha-bromopalmitate) down-regulate apo A-I and induce acyl-CoA oxidase gene expression through a direct transcriptional action on the hepatocyte. In contrast, only fenofibric acid, but not the other fibrates or fatty acids tested, decrease apo A-II gene expression in vitro.

Transgenic rabbits expressing human apolipoprotein A-I in the liver.

Human apolipoprotein A-I (apo A-I) transgenic rabbits were created by use of an 11-kb genomic human apo A-I construct containing a liver-specific promoter. Five independent transgenic lines were obtained in which human apo A-I gene had integrated and was expressed. Plasma levels of human apo A-I ranged from 8 to 100 mg/dL for the founder and up to 175 mg/dL for the progeny. Rabbit apo A-I levels were substantially decreased in the transgenic rabbits. HDL cholesterol (HDL-C) levels were higher in two of the five transgenic rabbit lines than in controls (line 20 versus nontransgenic littermate, HDL-C = 80 +/- 7 versus 37 +/- 6 mg/dL; line 8 versus nontransgenic littermate, HDL-C = 54 +/- 16 versus 35 +/- 6 mg/dL). This resulted in less atherogenic lipoprotein profiles, with very low (VLDL + LDL-C)/HDL-C ratios. HDL size and protein and lipid compositions were similar between transgenic and littermate nontransgenic rabbits. However, a large amount of pre-beta apo A-I-containing lipoproteins was observed in the plasma of the highest human apo A-I expressor. Cell cholesterol efflux was evaluated with the incubation of whole serum from transgenic and control rabbits. Cell cholesterol efflux was highly correlated with HDL cholesterol, with apo A-I, and with the presence of pre-beta apo A-I-containing lipoproteins. These rabbits will be an extremely useful model for the evaluation of the effect of increased hepatic apo A-I expression on atherosclerosis.