Expression of urokinase-type plasminogen activator receptor is increased during epileptogenesis in the rat hippocampus.

Urokinase-type plasminogen activator receptor (uPAR) is functionally a pleiotropic mediator involved in cell adhesion, proliferation, differentiation and migration as well as in matrix degradation, apoptosis, and angiogenesis in cancer tissue. Comparable cellular alterations occur in the brain during post-injury tissue repair. As the first step to assess the role of uPAR in brain tissue remodeling, we tested a hypothesis that uPAR expression is altered in the hippocampus during epilepsy-related circuitry reorganization. Epileptogenesis was triggered by inducing status epilepticus (SE) with electrical stimulation of the amygdala in rats. To monitor the development of SE and the occurrence of spontaneous seizures animals were continuously video-EEG monitored until sacrificed (1, 2, 4 or 14 days after SE). The hippocampal expression of uPAR was studied with real time qPCR and immunohistochemistry. Double-immunohistochemistry and confocal microscopy were used to investigate the expression of uPAR in astrocytes, microglia and neurons. We show that in the normal hippocampus the expression of uPAR was low and confined to small population of astrocytes and interneurons. In animals undergoing SE, uPAR expression increased dramatically, peaking at 1 and 4 days after SE. According to double-immunohistochemistry, uPAR was highly expressed in parvalbumin positive interneurons in the hippocampus and dentate gyrus, and in a subgroup of somatostatin and neuropeptide Y positive hilar interneurons. Increased uPAR expression during post-injury phase supports its contribution to tissue remodeling in the brain. Surviving hilar interneurons that are known to be denervated due to loss of afferent inputs in post-SE brain provide a target for future studies to investigate the contribution of uPAR in reinnervation of these cells, and to identify the signaling cascades that mediate the effects of uPAR.

Gene transfer using the mature form of VEGF-D reduces neointimal thickening through nitric oxide-dependent mechanism.

Gene transfer to the vessel wall using vascular endothelial growth factors (VEGFs) has shown therapeutic potential for the treatment of restenosis. In this study, we evaluated the effect of catheter-mediated adenoviral (Ad) gene transfer of the mature form of VEGF-D (VEGF-D(DeltaNDeltaC)) in balloon-denuded cholesterol-fed rabbit aorta. AdLacZ was used as a control. Transduced VEGF-D(DeltaNDeltaC) mRNA was detectable in the arterial wall with RT-PCR at 6, 14 and 28 days. Gene transfer efficiency as detected with X-gal staining 6 days after the AdLacZ transduction was 1.91 +/- 1.32% in intima. AdVEGF-D(DeltaNDeltaC) gene transfer led to 52% reduction in intima/media ratio (I/M) as compared to the AdLacZ controls at 14 days time point. At 6 days there were no differences in I/M, but the number of macrophages in the vessel wall was 85% lower in the AdVEGF-D(DeltaNDeltaC) group as compared to the controls. The therapeutic effect was no longer detectable 28 days after the gene transfer. The therapeutic effect of VEGF-D(DeltaNDeltaC) was nitric oxide (NO)-dependent as the feeding of NO synthase inhibitor, L-NAME, blocked the reduction in intimal thickening. It is concluded that AdVEGF-D(DeltaNDeltaC) gene transfer reduces intimal thickening and macrophage influx into the vessel wall in balloon-denuded rabbit aortas.

Angiographically guided utero-placental gene transfer in rabbits with adenoviruses, plasmid/liposomes and plasmid/polyethyleneimine complexes.

We examined the feasibility of gene transfer to rabbit placenta using adenoviruses, plasmid/liposomes and plasmid/polyethyleneimine (PEI) complexes. Pregnant New Zealand White rabbits (n = 17) were anesthetized and local gene transfer was done via a catheter inserted in uterine arteries under direct angiographic control. Either nuclear targeted LacZ adenoviruses (1.0 x 10(10) p.f.u.), nuclear targeted LacZ plasmid (500 microg)/liposome (DOTMA:DOPE 1:1) complexes or nuclear targeted LacZ plasmid (250 microg)/PEI (25 kDa) complexes (charge ratio +/-4) were used. Animals were killed 3 days later and detection of the transgene expression was done by X-gal staining and RT-PCR. Adenovirus-mediated gene transfer resulted in a high transfection efficiency (34 +/- 10%) in placental trophoplastic cells. Very little, if any, transfection was seen in fetal membranes. Plasmid/liposomes and plasmid/PEI complexes led to a very low (<0.01%) transfection efficiency in trophoblastic cells, but some transfection was seen in fetal membranes. A total of 25 fetuses were analyzed for the presence of transgene at the time of death. In most fetuses expression of the LacZ gene was below the sensitivity of the X-gal staining, but expression was detected by PCR in 50%, 50% and 42% of the analyzed fetuses after adenoviral, plasmid/PEI and plasmid/liposome gene transfer, respectively. No major inflammatory changes were present in the transfected placentas as analyzed by general histology and macrophage- and T cell-specific immunostainings. We conclude that catheter-mediated intravascular gene transfer with adenoviruses can be used for the transfection of placental trophoplastic cells, but plasmid complexes are inefficient for this purpose. However, selective angiographically guided gene transfer also led to leakage of the vector to fetuses. Therefore, if gene therapy is developed for the treatment of placental disorders, the gene-vector combination should not be harmful to the fetus and the expression of the transgene should only occur in placenta.

Optimized in situ PCR method for the detection of gene transfer vector in histological sections.

BACKGROUND: Detection of transferred genes in histological sections has been problematic due to low transfection efficiency and copy number achieved with current vectors. In situ polymerase chain reaction (in situ PCR) is a new method for the detection of low-abundance nucleic acid targets in tissue sections. METHODS: We have adapted in situ PCR method for the detection and histological localization of transgene DNA after in vivo and ex vivo retroviral gene transfer by using mild fixation and permeabilization methods. We used 4% paraformaldehyde/15% sucrose fixation combined with proteinase K permeabilization and microwave treatment. PCR signal was detected with non-radioactive digoxigenin-dUTP tailed oligonucleotide sense-probe. RESULTS: The method was applicable for both paraffin-embedded and frozen tissue sections and reached the sensitivity to detect a few copies of target DNA sequence per cell. CONCLUSIONS: In situ PCR is a sensitive method to localize integrated gene transfer vectors and to analyze the relationship between expression of the treatment gene and biological effects in the transfected tissues.

Intravascular adenovirus-mediated VEGF-C gene transfer reduces neointima formation in balloon-denuded rabbit aorta.

BACKGROUND: Gene transfer to the vessel wall may provide new possibilities for the treatment of vascular disorders, such as postangioplasty restenosis. In this study, we analyzed the effects of adenovirus-mediated vascular endothelial growth factor (VEGF)-C gene transfer on neointima formation after endothelial denudation in rabbits. For comparison, a second group was treated with VEGF-A adenovirus and a third group with lacZ adenovirus. Clinical-grade adenoviruses were used for the study. METHODS AND RESULTS: Aortas of cholesterol-fed New Zealand White rabbits were balloon-denuded, and gene transfer was performed 3 days later. Animals were euthanized 2 and 4 weeks after the gene transfer, and intima/media ratio (I/M), histology, and cell proliferation were analyzed. Two weeks after the gene transfer, I/M in the lacZ-transfected control group was 0. 57+/-0.04. VEGF-C gene transfer reduced I/M to 0.38+/-0.02 (P:<0.05 versus lacZ group). I/M in VEGF-A-treated animals was 0.49+/-0.17 (P:=NS). The tendency that both VEGF groups had smaller I/M persisted at the 4-week time point, when the lacZ group had an I/M of 0.73+/-0.16, the VEGF-C group 0.44+/-0.14, and the VEGF-A group 0. 63+/-0.21 (P:=NS). Expression of VEGF receptors 1, 2, and 3 was detected in the vessel wall by immunocytochemistry and in situ hybridization. As an additional control, the effect of adenovirus on cell proliferation was analyzed by performing gene transfer to intact aorta without endothelial denudation. No differences were seen in smooth muscle cell proliferation or I/M between lacZ adenovirus and 0.9% saline-treated animals. CONCLUSIONS: Adenovirus-mediated VEGF-C gene transfer may be useful for the treatment of postangioplasty restenosis and vessel wall thickening after vascular manipulations.

Biodistribution of adenoviral vector to nontarget tissues after local in vivo gene transfer to arterial wall using intravascular and periadventitial gene delivery methods.

Expression of transgene other than in the target tissue may cause side effects and safety problems in gene therapy. We analyzed biodistribution of transgene expression after intravascular and periadventitial gene delivery methods using the first generation nuclear-targeted lacZ adenovirus. RT-PCR and X-Gal stainings were used to study transgene expression 14 days after the gene transfer. After intravascular catheter-mediated gene transfer to rabbit aorta mimicking angioplasty procedure, the target vessel showed 1.1% +/- 0. 5 gene transfer efficiency. Other tissues showed varying lacZ gene expression indicating a systemic leakage of the vector with the highest transfection efficiency in hepatocytes (0.7% +/- 0.5). X-Gal staining of blood cells 24 h after the intravascular gene transfer indicated that a significant portion (1.8% +/- 0.8) of circulating monocytes was transfected. X-Gal-positive cells were also found in testis. After periadventitial gene transfer using a closed silicon capsule placed around the artery, 0.1% +/- 0.1 lacZ-positive cells were detected in the artery wall. Positive cells were also found in the liver and testis (<0.01%), indicating that the virus escapes even from the periadventitial space, although less extensively than during the intravascular application. We conclude that catheter-mediated intravascular and, to a lesser extent, periadventitial gene transfer lead to leakage of adenovirus to systemic circulation, followed by expression of the transgene in several tissues. Possible consequences of the ectopic expression of the transgene should be evaluated in gene therapy trials even if local gene delivery methods are used.

Baculovirus-mediated periadventitial gene transfer to rabbit carotid artery.

Recombinant Autographa californica multiple nuclear polyhedrosis viruses (AcMNPV) have recently been shown to transduce mammalian cells in vitro. Since baculoviruses offer many advantages over viruses currently used in gene therapy, we have tested them for in vivo gene transfer by constructing a baculovirus bearing a nuclear targeted beta-galactosidase marker gene (LacZ) under a CMV promoter. Both rabbit aortic smooth muscle cells (RAASMC) and human ECV-304 cells were susceptible to LacZ-baculovirus transduction. Transgene expression was evaluated in vivo by applying 1 x 10(9) p.f.u. of LacZ-baculoviruses or LacZ-adenoviruses in a silastic collar placed around rabbit carotid arteries in the absence of contact with blood components. As a result, baculoviruses led to transgene expression in adventitial cells in rabbit carotid arteries with efficiency comparable to adenoviruses. The beta-galactosidase gene expression was transient staying at a high level for 1 week but disappearing at the 14 day time-point. The arterial structure and endothelium remained intact in the baculovirus-transduced arteries, but macrophage-specific immunostaining detected signs of inflammation comparable to adenoviruses. Baculoviruses are thus able to mediate transient gene transfer in vivo and may become useful tools for gene therapy.

Insights into the molecular pathogenesis of atherosclerosis and therapeutic strategies using gene transfer.

Gene therapy for the treatment of atherosclerosis and related diseases has shown its potential in animal models and in the first human trials. Gene transfer to the vascular system can be performed both via intravascular and extravascular periadventitial routes. Intravascular gene transfer can be done with several types of catheters under fluoroscopic control. Extravascular gene transfer, on the other hand, provides a well-targeted gene delivery route available during vascular surgery. It can be done with direct injection or by using perivascular cuffs or surgical collagen sheets. Ex vivo gene delivery via transfected smooth muscle cells or endothelial cells might be useful for the production of secreted therapeutic compounds. Gene transfer to the liver has been used for the treatment of hyperlipidemia. The first clinical trials for the induction of therapeutic angiogenesis in ischemic myocardium or peripheral muscles with VEGF or FGF gene transfer are under way and preliminary results are promising. VEGF has also been used for the prevention of postangioplasty restenosis because of its capability to induce endothelial repair and production of NO and prostacyclin. However, further basic research is needed to fully understand the pathophysiological mechanisms involved in conditions related to atherosclerosis. Also, further development of gene transfer vectors and gene delivery techniques will improve the efficacy and safety of human gene therapy.

Efficient adventitial gene delivery to rabbit carotid artery with cationic polymer-plasmid complexes.

Different lipids and cationic polymers were tested in vitro for their ability to transfect rabbit aortic smooth muscle cells and human endothelial cells with lacZ marker gene. Toxicity of the complexes was evaluated with MTT assay. Selected plasmid-polymer complexes with different charge ratios were then tested for in vivo gene transfer efficiency using adventitial gene transfer by placing a silastic gene delivery reservoir (collar) around the carotid artery. Transfection efficiency was determined by X-gal staining 3 days after the gene transfer. Based on in vitro experiments, fractured polyamidoamine dendrimers and polyethylenimines (PEI) were selected for in vivo experiments. Fractured dendrimers (generation 6, +/- charge ratio of 3) had the highest in vivo gene transfer efficiency (4.4% +/- 1.7). PEI with molecular size of 25 kDa (+/- charge ratio 4) was also effective (2.8% +/- 1.8) in this model. PEI of 800 kDa showed a constant but modest gene transfer efficiency (1.8% +/- 0.1) with all charge ratios. A low level gene transfer was also detected with naked DNA (0.5% +/- 0.3). No signs of inflammation were seen in any of the study groups. We show here that in vitro cell culture experiments can be used to identify efficient in vivo gene transfer methods for arterial gene therapy, but the charge ratios for each complex must be optimized in vivo. It is concluded that fractured dendrimer and PEI are efficient gene delivery vehicles and can be used for arterial gene therapy via adventitial gene delivery route.

Gene therapy for angiogenesis, restenosis and related diseases.

Gene therapy may be useful for the treatment of atherosclerosis and related diseases. Gene transfer to vascular system can be performed both via intravascular and extravascular routes. Gene transfer to other tissues, such as liver and muscle, can also be used. The first clinical trials for the induction of therapeutic angiogenesis with VEGF gene transfer are under way, and preliminary results are promising. In the prevention of restenosis genes inhibiting cellular proliferation and increasing NO production, such as NOS and VEGF, have been used. However, more basic research is needed to fully understand pathophysiological mechanisms involved in conditions related to atherosclerosis. Also, further developments in gene transfer vectors and gene delivery techniques are required for the improvement of the efficacy of gene therapy.

Gene delivery to rabbit arteries using the collar model.

Emerging knowledge of molecular pathology of vascular diseases provides new targets for vascular gene therapy. Sufficient expression of a gene of interest in the vessel wall can be achieved using either extravascular or intravascular gene delivery approaches (1). Plasmid DNA, transferred by an extravascular approach, gives transfection efficiency high enough to cause biological effects. Plasmids and adenoviruses can be used for both extravascular and intravascular gene therapy. The collar model allows one to use controlled gene transfer. In this chapter we describe two gene delivery methods used for extravascular and intravascular gene transfer experiments.