Aorta of ApoE-deficient mice responds to atherogenic stimuli by a prelesional increase and subsequent decrease in the expression of antioxidant enzymes.

Oxidative stress has been implicated in the development of atherosclerotic lesions. We evaluated the relationship between extent of atherosclerotic lesion formation and vascular expression of pro- and antioxidant enzymes in apoE-deficient mice. On normal chow, these mice showed elevated serum cholesterol levels (7.5- to 9.5-fold), and age-dependent, spontaneous development of all stages of atherosclerotic lesions, starting at the age of 12 weeks. RNA was extracted from the aortic arch and descending aorta, and mRNA expression of pro- and antioxidant enzymes was measured with real-time PCR. Local infiltration of monocytes/macrophages, reflected by increased vascular expression of CD68 mRNA (>10-fold), indicated that the arch was more susceptible than the descending aorta. The expression of catalase-1 and various isoforms of superoxide dismutase, glutathione peroxidase, and glutathione S-transferase alpha was significantly increased in the aortic arch, but not in the descending aorta, in the period preceding lesion formation (age 6 to 12 weeks). These expression levels were 1.5 to 5 times higher than in age-matched wild-type animals. Remarkably, there was an inverse relationship between extent of lesion formation and the mRNA levels of antioxidant enzymes, most of which started to decline after 12 weeks, as lesions developed. In contrast, inducible nitric oxide synthase expression increased 4-fold in the aortic arch over the course of the disease. Our results suggest that the arterial wall responds to increased serum levels of atherogenic lipoproteins by stimulating expression of antioxidant enzymes. The observed co-ordinate decline in expression of many of these protective systems may greatly accelerate the development of atherosclerosis.

bis-Cholesteryl-conjugated phosphorothioate oligodeoxynucleotides are highly selectively taken up by the liver.

We previously modulated, by conjugating a single cholesterol, plasma protein binding and liver cell uptake of a phosphorothioate oligodeoxynucleotide (PS-ODN). In this study, we investigated the biological fate of a PS-ODN, denoted ISIS-9389 (3',5'-bis-cholesteryl-conjugated ISIS 3082), provided with two cholesteryl moieties. After intravenous injection of into rats, [(3)H]ISIS-9389 was cleared from plasma with a half-life of 23.6 +/- 0.3 min. After 90 min (approximately 95% cleared), the liver contained 83.0 +/- 0.8% of the dose. Spleen and bone (marrow), which constitute with the liver the reticuloendothelial system, contained 3.1 +/- 0.3 and 4.3 +/- 0.2%, respectively. All other tissues accumulated together <5% of the dose. The hepatic uptake of [(3)H]ISIS-9389 occurred mainly by endothelial cells (51.9 +/- 6.4% of the liver uptake). Parenchymal and Kupffer cells were responsible for 24.9 +/- 7.7 and 23.3 +/- 2.5%, respectively. Preinjected polyinosinic acid and polyadenylic acid reduced hepatic uptake, albeit the latter was less effective. This finding suggests implication of (multiple) scavenger receptors in liver uptake of ISIS-9389. The interaction of ISIS-9389 with plasma proteins, analyzed by size exclusion chromatography, differs from that of unconjugated PS-ODN and PS-ODN with a single cholesterol. Plasma-incubated ISIS-9389 was mainly recovered as a high molecular weight complex. In conclusion, conjugation of PS-ODNs with two cholesteryl moieties results in almost quantitative uptake by the liver. The liver targeting exceeds the already impressive gain in liver uptake achieved by conjugation of a single cholesterol, and is expected to increase the therapeutic activity against liver-associated targets and reduce side effects in nonhepatic tissues.

Induction of glutathione-S-transferase mRNA levels by chemopreventive selenocysteine Se-conjugates.

Several selenocysteine Se-conjugates (SeCys-conjugates) prevent against chemically induced carcinogenesis. Bioactivation to selenols (RSeH) by beta-lyases is thought to be critical, but the mechanism of tumor suppression remains unclear. Induction of phase II biotransformation enzymes is a possible mechanism of chemoprevention. In this study, we evaluated the isoform-selective induction of glutathione-S-transferase (GST) at the mRNA level using a quantitative reverse transcriptase polymerase chain reaction assay. In cultured primary rat hepatocytes and H35 Reuber rat hepatoma cells, SeCys-conjugates time-dependently increased mRNA levels of GST Alpha isoforms and GST Pi, but not of GST Mu isoforms. Se-allyl-L-selenocysteine, the most potent chemopreventive SeCys-conjugate so far known, was also the most active GST inducer. After exposure for 24hr, it elevated GSTA2, GSTA3, GSTA5, and GSTP mRNA levels in primary hepatocytes 3.2+/-0.4-, 1.9+/-0.1-, 4.3+/-0.3-, and 2.9+/-0.3-fold, respectively. Se-allyl-D-selenocysteine was significantly less active, suggesting that stereoselective conversion of SeCys-conjugates to selenols is involved in GST induction. In H35 Reuber hepatoma cells, where conversion of SeCys-conjugates to selenols was 2-6-fold lower than in primary hepatocytes, GST induction was also much lower than in primary hepatocytes. SeCys-conjugates did not induce cytochrome P450 1A1, 2B1/2, or 3A1. This indicates that SeCys-conjugates are monofunctional inducers of phase II biotransformation enzymes. The present results suggest that induction of GST expression contributes to the chemopreventive activity of SeCys-conjugates.

Selection of effective antisense oligodeoxynucleotides with a green fluorescent protein-based assay. Discovery of selective and potent inhibitors of glutathione S-transferase Mu expression.

Antisense oligodeoxynucleotides (AS-ODNs) are frequently used for the down-regulation of protein expression. Because the majority of potential antisense sequences lacks effectiveness, fast screening methods for the selection of effective AS-ODNs are needed. We describe a new cellular screening assay for the evaluation of the potency and specificity of new antisense sequences. Fusion constructs of the gene of interest and the gene encoding the enhanced green fluorescent protein (EGFP) are cotransfected with AS-ODNs to COS-7 cells. Subsequently, cells are analysed for expression of the EGFP fusion protein by flow cytometry. With the assay, we tested the effectiveness of a set of 15 phosphorothioate ODNs against rat glutathione S-transferase Mu1 (GSTM1) and/or Mu2 (GSTM2). We found several AS-ODNs that demonstrated potent, sequence-specific, and concentration-dependent inhibition of fusion protein expression. At 0.5 microm, AS-6 and AS-8 inhibited EGFP-GSTM1 expression by 95 +/- 4% and 81 +/- 6%, respectively. AS-5 and AS-10 were selective for GSTM2 (82 +/- 4% and 85 +/- 0.4% decrease, respectively). AS-2 and AS-3, targeted at homologous regions in GSTM1 and GSTM2, inhibited both isoforms (77-95% decrease). Other AS-ODNs were not effective or displayed non-target-specific inhibition of protein expression. The observed decrease in EGFP expression was accompanied by a decrease in GSTM enzyme activity. As isoform-selective, chemical inhibitors of GSTM and GSTM knock-out mice are presently unavailable, the selected AS-ODNs constitute important tools for the study of the role of GSTM in detoxification of xenobiotics and protection against chemical-induced carcinogenesis.

Design of a targeted peptide nucleic acid prodrug to inhibit hepatic human microsomal triglyceride transfer protein expression in hepatocytes.

In this study, we present the design and synthesis of an antisense peptide nucleic acid (asPNA) prodrug, which displays an improved biodistribution profile and an equally improved capacity to reduce the levels of target mRNA. The prodrug, K(GalNAc)(2)-asPNA, comprised of a 14-mer sequence complementary to the human microsomal triglyceride transfer protein (huMTP) gene, conjugated to a high-affinity tag for the hepatic asialoglycoprotein receptor (K(GalNAc)(2)). The prodrug was avidly bound and rapidly internalized by HepG2s. After iv injection into mice, K(GalNAc)(2)-asPNA accumulated in the parenchymal liver cells to a much greater extent than nonconjugated PNA (46% +/- 1% vs 3.1% +/- 0.5% of the injected dose, respectively). The prodrug was able to reduce MTP mRNA levels in HepG2 cells by 35-40% (P < 0.02) at 100 nM in an asialoglycoprotein receptor- and sequence-dependent fashion. In conclusion, hepatocyte-targeted PNA prodrugs combine a greatly improved tropism with an enhanced local intracellular availability and activity, making them attractive therapeutics to lower the expression level of hepatic target genes such as MTP.

Selection of antisense oligodeoxynucleotides against glutathione S-transferase Mu.

The aim of the present study was to identify functional antisense oligodeoxynucleotides (ODNs) against the rat glutathione S-transferase Mu (GSTM) isoforms, GSTM1 and GSTM2. These antisense ODNs would enable the study of the physiological consequences of GSTM deficiency. Because it has been suggested that the effectiveness of antisense ODNs is dependent on the secondary mRNA structures of their target sites, we made mRNA secondary structure predictions with two software packages, Mfold and STAR. The two programs produced only marginally similar structures, which can probably be attributed to differences in the algorithms used. The effectiveness of a set of 18 antisense ODNs was evaluated with a cell-free transcription/translation assay, and their activity was correlated with the predicted secondary RNA structures. Four phosphodiester ODNs specific for GSTM1, two ODNs specific for GSTM2, and four ODNs targeted at both GSTM isoforms were found to be potent, sequence-specific, and RNase H-dependent inhibitors of protein expression. The IC50 value of the most potent ODN was approximately 100 nM. Antisense ODNs targeted against regions that were predicted by STAR to be predominantly single stranded were more potent than antisense ODNs against double-stranded regions. Such a correlation was not found for the Mfold prediction. Our data suggest that simulation of the local folding of RNA facilitates the discovery of potent antisense sequences. In conclusion, we selected several promising antisense sequences, which, when synthesized as biologically stable oligonucleotides, can be applied for study of the physiological impact of reduced GSTM expression.