Long-term follow-up of porcine male germ cells transplanted into mouse testes.

This study investigated the effect of increased phylogenetic distance on the outcome of spermatogonial transplantation, with porcine donors and mice recipients. It was designed to develop a technique for detecting foreign donor cells in recipient animals. Porcine male germ cells were harvested from postnatal male testes and incubated with the lipophilic membrane dye PKH-26. For transplantation, approximately 10(6) PKH-26-labelled porcine male germ cells were injected into the efferent ducts of mouse testes. Animals were sacrificed at post-graft days 1, 10, 30, 45, 60 and 150 (n = 5 each). Serial frozen sections of explanted testes were prepared for detecting labelled cells. Transplanted porcine donor cells were easily detected in the recipient tubules for 8 weeks. After transplantation, we could detect both incorporation into the basement membrane and differentiation of grafted porcine donor cells by our double detection system, using PKH staining and slide PCR. However, our RT-PCR and apoptosis results revealed that most of the grafted porcine male donor cells could not differentiate past early-meiotic spermatocytes. We could induce partial differentiation of xenografted porcine donor cells in mouse testes, but not full induction of spermatogenesis. We have developed a very reliable technique for detecting foreign donor cells in recipient animals using a combination of PKH staining and slide PCR methods. Our results provide a valuable experimental model for applying and evaluating this technology in other species.

E2F decoy oligodeoxynucleotides on neointimal hyperplasia in canine vein graft.

Double-stranded DNA with high affinity to E2F as a decoy cis-element blocks the activation of genes mediating the cell cycle, resulting in effective suppression of the smooth muscle cell proliferation that causes intimal hyperplasia. To evaluate the effect of the E2F decoy to suppress neointimal hyperplasia autogenous venous bypass grafts were performed in dogs after incubation with heparin (group 1), with E2F decoy oligodeoxynucleotides (ODN) (groups 2 and 3), or with a random ODN (group 4) using a Japan-liposomeal method based on a hemagglutinating virus. The intimal and medial cross-sectional surface area of the anastomotic site was measured at 4 months after bypass surgery in groups 1, 3, and 4 by computerized planimetry and at 4 weeks in group 2 to compare the intimal/medial (I/M) area ratios. Autogenous vein grafts treated with E2F decoy showed a significant reduction in I/M area ratio (0.26 +/- 0.11) compared with the heparin-treated control group (1.49 +/- 0.29) or the mismatched ODN-treated group (1.61 +/- 0.28; P = .000). There was no difference in the I/M area ratio according to experimental periods (groups 2 vs 3: 0.26 +/- 0.11 vs 0.37 +/- 0.32; P = .446) or the anastomotic sites (proximal vs distal; P = .934). In conclusion, an E2F decoy can suppress neointimal hyperplasia in autogenous vein grafts, which may prolong patency by reducing graft stenosis.

The effects of the overexpression of recombinant uncoupling protein 2 on proliferation, migration and plasminogen activator inhibitor 1 expression in human vascular smooth muscle cells.

AIMS/HYPOTHESIS: Increased oxidative stress in vascular smooth muscle cells (VSMCs) has been implicated in the pathogenesis of accelerated atherosclerosis in patients with diabetes mellitus. Uncoupling protein 2 (UCP-2) is an important regulator of intracellular reactive oxygen species (ROS) production. We hypothesised that UCP-2 functions as an inhibitor of the atherosclerotic process in VSMCs. METHODS: Overexpression of human UCP-2 was performed in primary cultured human VSMCs (HVSMCs) via adenovirus-mediated gene transfer. Its effects on ROS production, AP-1 activity, plasminogen activator inhibitor 1 (PAI-1) gene expression, and cellular proliferation and migration were measured in response to high glucose and angiotensin II (Ang II) concentrations, two major factors in the pathogenesis of atherosclerosis in patients with diabetes and hypertension. Mitochondrial membrane potential and NAD(P)H oxidase activity were also measured. RESULTS: High glucose and Ang II caused transient mitochondrial membrane hyperpolarisation. They also significantly stimulated ROS production, NAD(P)H oxidase activity, mitochondrial membrane potential, AP-1 activity, PAI-1 mRNA expression, and proliferation and migration of HVSMCs. Adenovirus-mediated transfer of the UCP-2 gene reversed all of these effects. CONCLUSIONS/INTERPRETATION: The present study demonstrates that UCP-2 can modify atherosclerotic processes in HVSMCs in response to high glucose and Ang II. Our data suggest that agents increasing UCP-2 expression in vascular cells may help prevent the development and progression of atherosclerosis in patients with diabetes and hypertension.

Transcription factor decoy for AP-1 reduces mesangial cell proliferation and extracellular matrix production in vitro and in vivo.

Diabetic nephropathy is characterized by an expansion of glomerular mesangium, caused by mesangial cell proliferation and excessive accumulation of extracellular matrix (ECM) proteins, which eventually leads to glomerulosclerosis and renal failure. Activator protein-1 (AP-1), a transcription factor, is implicated in the transcriptional regulation of a wide range of genes participating in cell proliferation and ECM production. This investigation was undertaken to test the hypothesis that AP-1 plays an important role in ECM gene expression, and to develop a molecular therapeutic strategy based on decoy oligodeoxynucleotides (ODN). In this report, we show that transfection with AP-1 decoy ODN strongly inhibits high glucose- and angiotensin II-induced cell proliferation and expression of ECM genes in cultured mesangial cells in vitro. Administration of AP-1 decoy ODN into streptozotocin-induced diabetic rat kidney in vivo using the hemagglutinating virus of Japan (HVJ)-liposome method virtually abolished TGF-beta1 and plasminogen activator inhibitor-1 expression. Our results collectively indicate that AP-1 activation is crucial for mesangial cell proliferation and ECM production in response to high glucose and angiotensin II. Moreover, use of stable AP-1 decoy ODN combined with the highly effective HVJ-liposome method provides a novel potential molecular therapeutic strategy for the prevention of diabetic nephropathy.

Advantages of the circular dumbbell decoy in gene therapy and studies of gene regulation.

Decoy oligodeoxynucleotides (ODN) that can reduce the trans-activity of transcription factors may be highly useful in gene therapy and the study of transcriptional regulation. Several different types of these double-stranded DNA decoys have been developed, including unmodified oligonucleotide duplexes, alphabeta-anomeric oligonucleotides, and oligonucleotide duplexes with methylphosphonate- and phosphorothioate-modified linkages. The latter ODNs have been particularly extensively studied but suffer from a number of limitations, including their insensitivity to polymerases, their lack of sequence specificity, and their tendency to activate immune responses. To resolve these problems, circular dumbbell (CD) double-stranded ODNs were developed. These CD ODNs are constructed by the circularization of the 3' and 5' ends of the oligonucleotides and enzymatic ligation. They exhibit high resistance to nucleases, are easily taken up by cells, and have a nontoxic unmodified backbone that resembles natural DNA. In this article, we review the method of constructing CD ODNs and their advantages compared to other modified ODNs for use as transcription decoys.

Novel E2F decoy oligodeoxynucleotides inhibit in vitro vascular smooth muscle cell proliferation and in vivo neointimal hyperplasia.

The transcription factor, E2F, plays a critical role in the trans-activation of several genes involved in cell cycle regulation. Previous studies showed that the transfection of cis element double-stranded decoy oligodeoxynucleotides (ODNs) corresponding to E2F binding sites inhibited the proliferation of vascular smooth muscle cells (VSMCs) and neointimal hyperplasia in injured vessels. We have developed a novel E2F decoy ODN with a circular dumbbell structure (CD-E2F) and compared its effects with those of the conventional phosphorothioated E2F decoy (PS-E2F) ODN. CD-E2F ODN was more stable than PS-E2F ODN, largely preserving its structural integrity after incubation in the presence of nucleases and sera. Moreover, CD-E2F ODN inhibited high glucose- and serum-induced transcriptional expression of cell cycle regulatory genes more strongly than PS-E2F ODN. Transfection of CD-E2F ODN resulted in more effective inhibition of VSMC proliferation in vitro and neointimal formation in vivo, compared with PS-E2F ODN. An approximately 40-50% lower dose of CD-E2F ODN than PS-E2F ODN was sufficient to attain similar effects. In conclusion, our results indicate that CD-E2F ODN may be a valuable tool in gene therapy protocols for inhibiting VSMC proliferation and studying transcriptional regulation.

Transcription factor decoy for activator protein-1 (AP-1) inhibits high glucose- and angiotensin II-induced type 1 plasminogen activator inhibitor (PAI-1) gene expression in cultured human vascular smooth muscle cells.

AIMS/HYPOTHESIS: Multiple factors, including hyperglycaemia and angiotensin II (Ang II), stimulate plasminogen activator inhibitor-1 (PAI-1) gene expression in human vascular smooth muscle cells. This study tested the hypothesis that hyperglycaemia and Ang II stimulate PAI-1 gene expression through activator protein-1 (AP-1) binding sites. METHODS: We evaluated the role of AP-1 in PAI-1 gene expression in human vascular smooth muscle cells under high D-glucose and Ang II stimulation using a double-stranded cis-element AP-1 oligodeoxynucleotide (decoy ODN). RESULTS: Activator protein 1 activity was stimulated by high glucose and Ang II treatment and the AP-1 decoy ODN, but not a mismatched decoy ODN, competed for AP-1 activity. The increase in PAI-1 expression by high glucose and Ang II was significantly attenuated by the AP-1 decoy ODN (p <0.05 or p < 0.01). The increase in PAI-1 expression by high glucose and Ang II action on AP-1 sites was also confirmed by promoter analysis of PAI-1. Activator protein 1 activation in response to either high glucose or co-stimulation with high glucose and Ang II was inhibited completely by calphostin C (a PKC inhibitor) and partially by genistein (a protein tyrosine kinase inhibitor). CONCLUSION/INTERPRETATION: This study shows that high glucose and Ang II stimulate PAI-1 expression through AP-1 binding sites. Signal transduction after AP-1 activation by both high glucose and Ang II largely depends on PKC activation. These data indicate an important role for AP-1 in PAI-1 expression.

Nucleotide sequence and differential expression of the human 3-phosphoglycerate dehydrogenase gene.

The nucleotide sequence of Hs 3-PGDH gene, encoding human 3-phosphoglycerate dehydrogenase that catalyzes the initiating step in the phosphorylated pathway of serine biosynthesis, has been determined. The 3-PGDH gene has a predicted 533 amino acid open reading frame, encoding a 56.8kDa protein that shares 94.0% similarity with rat-liver 3-PGDH. Two different transcripts corresponding to 3-PGDH mRNA were detected in human normal tissues. A dominant 2.1kb transcript was expressed at high levels in prostate, testis, ovary, brain, liver, kidney, and pancreas, and weakly expressed in thymus, colon, and heart. A 710bp transcript also appeared as a weaker band where the 2.1kb mRNA was expressed, and it was more significant than the 2.1kb mRNA in heart and skeletal muscle. The TPA-induced monocytic differentiation of U937, which also resulted in growth arrest, abruptly downregulated the expression of 3-PGDH. Removal of TPA restored cell growth through the retrodifferentiation process and subsequent expression of 3-PGDH. The 3-PGDH mRNA was markedly expressed in human leukemias, lymphoma Sup-T1, colon adenocarcinoma COLO 320DM, epitheloid carcinoma HeLa S3, and murine lymphoma BW5147.G.1.4, but not in human leukemia K562. This report demonstrates that the human 3-PGDH gene is regulated at the transcriptional level depending on tissue specificty and cellular proliferative status, and its transcriptional regulation mechanism may be a useful target for diagnosis and therapy of cancer.