The transcription factor, E2F, plays a critical role in the transactivation 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.

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.

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.