Search

Neointimal formation is a common feature after angioplasty, bypass grafting and stenting. Angioplasty damages endothelium, causing pathological changes in arteries which lead to smooth muscle cell proliferation, synthesis of extracellular matrix components and eventually restenosis formation. Adenoviruses offer an efficient transgene expression in the vascular system. In this study, we compared the effects of different gene combinations. We wanted to find out whether adenoviral catheter-mediated delivery of an additive combination of the vascular endothelial growth factor (VEGF)-A with VEGF-C is more effective than the combination of tissue inhibitor of metalloproteinase 1 (TIMP-1) alone or with VEGF-C in a rabbit balloon denudation model. Additionally, we wanted to clarify whether the combination therapy prolongs the treatment effect. It was found that TIMP-1 alone prevents restenosis and that the combination of VEGF-A and VEGF-C has a similar effect at the 2-week time point. However, the combination of VEGF-A and VEGF-C lost the treatment effect at the 4-week time point due to the catch-up growth of neointima. On the other hand, TIMP-1 and the combination of TIMP-1 with VEGF-C still had an extended treatment effect at the 4-week time point. When considering the gene combination used in this study, it is concluded that gene therapy with adenoviral TIMP-1 alone is sufficient in reducing restenosis and that combination gene therapy does not bring any significant advantages., (Copyright (c) 2005 S. Karger AG, Basel.)

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.

Postangioplasty restenosis is a multifactorial process and involves mechanisms such as inflammation and stimulation of the expression of growth factors. Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) can modify inflammatory responses by hydrolyzing phospholipids with shortened and/or oxidized sn-2 residues. In this study, we tested a hypothesis that adenovirus-mediated Lp-PLA(2) gene transfer can reduce restenosis in rabbits. Aortas of cholesterol-fed NZW rabbits were balloon-denuded and intra-arterial gene transfer was performed using Dispatch catheter with Lp-PLA(2) or LacZ adenoviruses (1.15 x 10(10)pfu). Intima/media ratio (I/M), histology and cell proliferation were analyzed. Two weeks after the gene transfer I/M in the LacZ-transduced control group was 0.45+/-0.05 but Lp-PLA(2) gene transfer reduced I/M to 0.25+/-0.03. At four weeks time point I/M in the Lp-PLA(2) group (0.34+/-0.05) was also lower than in the LacZ group (0.53+/-0.06). Plasma Lp-PLA(2) activity was increased in the Lp-PLA(2) group (48.2+/-4.2) as compared to the LacZ group (33.6+/-3.51) at two weeks time point. Transgene expression was detected in the arterial wall two and four weeks after the procedure. Apoptosis was higher in the control vessels than in the Lp-PLA(2) group at two weeks time point. In conclusion, local adenovirus-mediated Lp-PLA(2) gene transfer resulted in a significant reduction in neointima formation in balloon-denuded rabbit aorta and may be useful for the prevention of restenosis after arterial manipulations.

Background: Platelet-derived growth factor (PDGF) antagonists have demonstrated beneficial effects on neointima formation, but in studies using PDGF inhibitors and extended follow-up, the lesions reoccur. These findings implicate a need to combine targeting of PDGF with other strategies. Stimulation of reendothelialization by treatment with endothelial cell mitogens of the vascular endothelial growth factor (VEGF) family counteracts restenosis, but there are also concerns regarding the durability of the effect with this approach., Methods and Results: To explore whether a combined use of PDGF antagonist and stimulation of reendothelialization confers better results than each therapy alone, we combined systemic administration of imatinib mesylate (STI571/Gleevec, 10 mg/kg(-1) per d(-1)), a tyrosine kinase inhibitor with activity against PDGF receptors, with local intravascular adenovirus-mediated VEGF-C gene transfer (1.15x10(10) pfu) in cholesterol-fed, balloon-injured rabbits. Throughout the course of the STI571 therapy, the circulating concentrations were able to suppress PDGF receptor phosphorylation. At 3 weeks, the treatment with STI571 led to a transient decrease in intralesion macrophages and to an increase in intimal smooth muscle cell apoptosis. VEGF-C application reduced neointima formation and accelerated reendothelialization. However, none of the therapies alone reduced intimal thickening at a 6-week time point, whereas the combined treatment led to a persistent reduction (55% versus control) in lesion size at this time point., Conclusions: Our study provides one of the first successful examples of gene therapy combined with a pharmacological treatment to modulate 2 distinct ligand-receptor signaling systems and suggests combination of local VEGF-C gene therapy with systemic inhibition of PDGF signaling as a novel principle to prevent intimal hyperplasia after vascular manipulations.

We have developed an ex vivo gene transfer technique to rabbit arterial wall using autologous smooth muscle cells (SMCs). SMCs were harvested from rabbit ear artery, transduced in vitro with vesicular stomatitis virus G-glycoprotein pseudotyped retrovirus or feline immunodeficiency virus (FIV) and returned to the adventitial surface of the carotid artery using a periadventitial silicone collar or collagen sheet placed around the artery. Beta-galactosidase (lacZ) and human apolipoprotein E3 (apoE3) cDNAs were used as transgenes. After retrovirus-mediated gene transfer of lacZ the selected cells implanted with high efficiency and expressed lacZ marker gene at a very high level 7 and 14 days after the operation. The level of lacZ expression decreased thereafter but was still detectable 12 weeks after the gene transfer, and was exclusively localized to the site of cell implantation inside the collar. Utilizing FIV vector expressing apoE3, low levels of apoE were measured from serum collected from a low-density lipoprotein receptor deficient Watanabe heritable hyperlipidemic rabbits 1 month after the gene transfer. The physiological effect of apoE expression was detected as transiently elevated serum cholesterol levels. The results indicate that the model can be used for high efficiency local gene transfer in arteries, e.g. during vascular surgery. The model is also valuable for studying expression, stability and safety of new gene transfer vectors and their expression products in vivo.

Objective: Vascular endothelial growth factor-D (VEGF-D) is a recently characterized member of the VEGF family, but its expression in atherosclerotic lesions remains unknown. We studied the expression of VEGF-D and its receptors (VEGFR-2 and VEGFR-3) in normal and atherosclerotic human arteries, and compared that to the expression pattern of VEGF-A., Methods: Human arterial samples (n=39) obtained from amputation operations and fast autopsies were classified according to the stage of atherosclerosis and studied by immunohistochemistry. The results were confirmed by in situ hybridization and RT-PCR., Results: We found that while VEGF-A expression increased during atherogenesis, VEGF-D expression remained relatively stable only decreasing in complicated lesions. In normal arteries and in early lesions VEGF-D was mainly expressed in smooth muscle cells, whereas in complicated atherosclerotic lesions the expression was most prominent in macrophages and also colocalized with plaque neovascularization. By comparing the staining profiles of different antibodies, we found that proteolytic processing of VEGF-D was efficient in the vessel wall. VEGFR-2, but not VEGFR-3, was expressed in the vessel wall at every stage of atherosclerosis., Conclusions: Our results suggest that in large arteries VEGF-D is mainly expressed in smooth muscle cells and that it may have a role in the maintenance of vascular homeostasis. However, in complicated lesions it was also expressed in macrophages and may contribute to plaque neovascularization. The constitutive expression of VEGFR-2 in arteries suggests that it may be one of the principal mediators of the VEGF-D effects in large arteries.

DNA methylation is a form of epigenetic modification of the genome that can regulate gene expression. Hypermethylation of CpG islands in the promoter areas leads to decreased gene expression, whereas promoters of actively transcribed genes remain nonmethylated. Because of cellular proliferation and monoclonality of at least some of the lesion cells, atherosclerotic lesions have been compared with benign vascular tumors.1,2 However, although genetic and epigenetic background favors neoplastic transformation, atherosclerotic plaques never develop to malignant tumors. Among cancer cells, common features are genome-wide hypomethylation, which correlates with transformation and tumor progression. Recent studies have shown that DNA methylation changes occur also during atherogenesis and may contribute to the lesion development.

Osteoporosis is a major problem in elderly population. We tested the hypothesis whether vascular endothelial growth factor (VEGF-A) gene transfer is an appropriate way to enhance bone formation and recruitment of osteoblasts in vivo. Adenovirus vectors containing VEGF-A or lacZ cDNAs (1.4x10(10) pfu) were injected locally into right distal femurs of New Zealand White rabbits. Saline was injected into all contralateral distal femurs. One and three weeks after the gene transfers femurs were collected for analyses. X-Gal staining showed that up to 20% of the bone marrow cells were transfected although gene transfer also resulted in biodistribution of the vector and expression of the transgene in liver and spleen. Trabecular bone hard tissue histomorphometry of the distal femurs was performed to analyze the effect of gene transfer on bone turnover. When compared with unilateral lacZ transfected trabecular bone at one-week and three-week time points, VEGF-A gene transfer significantly increased bone formation parameters, such as osteoblast number, osteoid volume, and bone volume. Also, bone resorption surface was greatly reduced. It is concluded that injection of adenovirus vector can transfect bone marrow cells in vivo with a relatively high efficiency. Our results suggest that adenovirus-mediated VEGF-A gene transfer induces bone formation via increasing osteoblast activity and may be useful for the treatment of osteoporosis and other diseases that require efficient osteogenic therapy.

A better understanding of atherogenesis at the level of gene expression could lead to the identification of new therapeutic strategies for vascular diseases. With DNA array technology, it is possible to identify multiple, simultaneous changes in gene expression in small tissue samples from atherosclerotic arteries. We analyzed gene expression in normal arteries and in immunohistologically characterized human advanced atherosclerotic lesions using an array of 18376 cDNA fragments. The array method was first validated by detecting a group of genes (n=17) that were already known to be connected to atherogenesis. These genes included e.g. Apolipoprotein E, CD68, TIMP and phospholipase D. Next we detected 75 differentially expressed genes that were previously not connected to atherogenesis. A subgroup of genes involved in cell signaling and proliferation was selected for further analyzes with in situ hybridization and RT-PCR which confirmed array results by showing induction in advanced lesions of Janus kinase 1 (JAK-1) which is an important signaling molecule in activated macrophages; VEGF receptor-2 which mediates angiogenic and vasculoprotective effects of VEGF; and an unknown gene, which mapped on chromosome 19. It is concluded that DNA array technology enables fast screening of gene expression in small samples of atherosclerotic lesions. The technique will be useful for the identification of new factors, such as JAK-1 and VEGF receptor-2, which may play an important role in atherogenesis.

In this study we have attached cyclic targeting peptides by way of a poly-lysine spacer on the surface of an adenovirus using a transglutaminase enzymatic reaction to enhance transduction efficiency and to modify tissue tropism in vivo. Nuclear targeted lacZ- and TIMP-1-encoding adenoviruses were coupled to a peptide-motif (HWGF) that can bind to matrix metalloproteinase (MMP)-2 and MMP-9. Modified viruses were used to evaluate gene transfer efficiency, biodistribution, and the effect on neointima formation following balloon denudation injury. In vitro, both rabbit aortic smooth muscle cells and human endothelial hybridoma cells demonstrated significantly increased reporter gene expression with HWGF-modified adenoviruses (AdlacZ(HWGF)) compared with control (AdlacZ) or mismatch peptide-modified (AdlacZ(MM)) adenoviruses. However, in human hepatocellular Hep-G2 cells, both AdlacZ(HWGF) and AdlacZ(MM) produced significantly lower transgene expression compared with the respective control viruses. In vivo, local intravascular catheter-mediated gene transfer of a HWGF-targeted TIMP-1-encoding adenovirus (AdTIMP-1(HWGF)) significantly reduced intimal thickening in a rabbit aortic balloon denudation model (P < 0.05) compared with the control adenovirus. X-Gal staining and biodistribution analyses with TaqMan RT-PCR revealed that the cyclic peptides altered vector tropism and, in particular, reduced transduction of the liver. We found that the HWGF peptide modification increased transduction efficiency of the adenovirus-mediated gene transfer in smooth muscle cells and endothelial cells in in vitro and enhanced gene transfer to the arterial wall in vivo; that peptide modification of adenoviruses beneficially modulated tissue tropism in vivo; and that efficient TIMP-1 gene transfer reduced intimal thickening in an established restenosis model in rabbits.

Background: Recent studies have suggested the therapeutic potential of vascular endothelial growth factor (VEGF) gene therapy in ischemic skeletal muscle. However, only limited information is available about the effects of VEGF gene therapy in different regions of ischemic limbs, effects of control adenoviruses, and biodistribution of the transgenes after intramuscular (i.m.) administration. Here we studied angiogenesis and side effects of adenovirus-mediated VEGF and beta-galactosidase (LacZ) gene transfers in ischemic rabbit hindlimbs., Methods and Results: Ten days after induction of ischemia, rabbits were treated with i.m. injections of saline, LacZ adenovirus (AdLacZ; 2x10(10) pfu) or adenovirus encoding mouse VEGF(164) (AdVEGF; 2x10(10) pfu). In rabbits treated with AdVEGF an increase in serum VEGF(164) levels was detected by ELISA three and seven days after the gene transfer. 30 days after the gene transfer a positive effect on capillary density was observed in the thigh region both in rabbits treated with AdVEGF and AdLacZ compared with animals that received saline. On the other hand, AdVEGF and AdLacZ gene transfers had no effect on the capillary density in the calf region on day 30. A positive correlation between the capillary density and the number of collateral arteries was observed in the thigh. Hindlimb and testis edema and excess non-physiological growth of capillaries were detected as adverse effects of the AdVEGF gene therapy. Biodistribution analysis showed that the transgene was present not only in the target muscle, but also in ectopic tissues seven days after i.m. gene transfer., Conclusions: The results suggest that a high dose of adenoviral vector encoding either AdVEGF or AdLacZ induces angiogenesis in the rabbit hindlimb ischemia model; i.m. injection of adenovirus leads to the transfection of ectopic organs; and AdVEGF gene transfer induces edema in ischemic skeletal muscle., (Copyright 2002 John Wiley & Sons, Ltd.)

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible endothelial cell mitogen and survival factor. Its receptor VEGFR-2 (KDR/Flk-1) mediates these effects. We studied the expression of VEGF and VEGFR-2 in ischemic human and rabbit skeletal muscle by immunohistochemistry and in situ hybridization. Human samples were obtained from eight lower limb amputations because of acute or chronic critical ischemia. In chronically ischemic human skeletal muscle VEGF and VEGFR-2 expression was restricted to atrophic and regenerating skeletal myocytes, whereas in acutely ischemic limbs VEGF and VEGFR-2 were expressed diffusely in the affected muscle. Hypoxia-inducible factor-1alpha was associated with VEGF and VEGFR-2 expression both in acute and chronic ischemia but not in regeneration. Hindlimb ischemia was induced in 20 New Zealand White rabbits by excising the femoral artery. Magnetic resonance imaging and histological sections revealed extensive ischemic damage in the thigh and leg muscles of ischemic rabbit hindlimbs with VEGF expression similar to acute human lower limb ischemia. After 1 and 3 weeks of ischemia VEGF expression was restricted to regenerating myotubes and by 6 weeks regeneration and expression of VEGF was diminished. VEGFR-2 expression was co-localized with VEGF expression in regenerating myotubes. Macrophages and an increased number of capillaries were associated with areas of ischemic muscle expressing VEGF and VEGFR-2. In conclusion, two patterns of VEGF and VEGFR-2 expression in human and rabbit ischemic skeletal muscle are demonstrated. In acute skeletal muscle ischemia VEGF and VEGFR-2 are expressed diffusely in the affected muscle. In chronic skeletal muscle ischemia and in skeletal muscle recovering from ischemia VEGF and VEGFR-2 expression are restricted to atrophic and regenerating muscle cells suggesting the operation of an autocrine pathway that may promote survival and regeneration of myocytes.

Arterial smooth muscle cell (SMC) migration and proliferation are central features in atherogenesis. Altered gene expression and cell proliferation in atherosclerotic lesions have some similar characteristics with certain solid tumors and thus might have similar mechanisms that lead to SMC proliferation. Among cancer cells common features are genome-wide hypomethylation which correlates with transformation and tumor progression, and coincident overexpression of methyltransferase (MTase). The purpose of the present study was to analyze whether alterations in DNA methylation and MTase expression are present in atherosclerotic lesions. A significant reduction in genomic 5-methylcytosine content was detected in advanced human atherosclerotic lesions and in lesions of ApoE knock-out mice. SMC were shown to develop hypomethylation in vitro during transformation from a contractile to synthetic phenotype. Balloon denudation of New Zealand White rabbit aorta caused proliferation of intimal SMC with concomitant genomic hypomethylation in the thickened intima. By using in situ hybridization the overall transcriptional activity was found to be increased in clusters of lesion SMC. Marked heterogeneity was seen in MTase mRNA expression in various types of atherosclerotic lesions among intimal and medial SMC. These findings show that (1) genomic hypomethylation occurs during atherogenesis in human, mouse and rabbit lesions and that it correlates with increased transcriptional activity; (2) MTase is expressed in atherosclerotic lesions; and (3) hypomethylation is present in advanced lesions at the same level as in malignant tumors and may affect cellular proliferation and gene expression in atherosclerotic lesions.

Local gene transfer into the vascular wall offers a promising alternative to treat atherosclerosis-related diseases. Blood vessels are among the easiest targets for gene therapy because of percutaneous, catheter-based treatment methods. On the other hand, gene transfer to the artery wall can also be accomplished from adventitia either by ex vivo gene transfer and implantation of transfected cells or by direct in vivo gene transfer methods. In the future, as the pathological processes in arteries are better understood, several therapeutic genes could be combined and these "gene cocktails" are expected to produce enhanced therapeutic effects in vascular gene therapy. We have developed a new, efficient technique for performing ex vivo gene transfer to rabbit arterial wall using autologous SMC. The cells were harvested from rabbit ear artery, transfected in vitro with VSV-G pseudotyped lacZ retrovirus, and returned back to the adventitial surface of the carotid artery using a silicone collar or collagen sheet placed around the artery. The transduced SMCs implanted with a high efficiency and expressed beta-galactosidase marker gene at a very high level 7 days and 14 days after the operation. The level of lacZ expression decreased thereafter, but was still easily detectable for at least 6 months and was exclusively localized to the site of cell implantation inside the collar. Development of new vectors, such as baculovirus, for gene transfer will provide targeted, efficient, and safer methods for gene delivery. Plasmids and viruses coding for more than one protein, and bearing regulatory elements, would be useful for future gene therapy applications. Also, constructing second-generation viruses that contain fewer endogenous genes in their genome may reduce immunological reactions caused by the first-generation adenoviruses. In conditions where stable expression of therapeutic proteins is needed, it is necessary to develop better ex vivo and in vivo gene transfer strategies. Also, production of viruses that can efficiently transfect nondividing cells will be important for future applications of vascular gene therapy. However, current knowledge from vascular gene transfer experiments strongly suggests that vascular gene transfer is a promising new alternative for the treatment of cardiovascular diseases.

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.

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.

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.

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.

Blood vessels are among the easiest targets for gene therapy. However, no data are available about the safety and feasibility of intracoronary gene transfer in humans. We studied the safety and efficacy of catheter-mediated vascular endothelial growth factor (VEGF) plasmid/liposome (P/L) gene transfer in human coronary arteries after percutaneous translumenal coronary angioplasty (PTCA) in a randomized, double-blinded, placebo-controlled study. The optimized angioplasty/gene delivery method was previously shown to lead to detectable VEGF gene expression in human peripheral arteries as analyzed from amputated leg samples. Gene transfer to coronary arteries was done with a perfusion-infusion catheter, using 1000 microg of VEGF or beta-galactosidase plasmid complexed with 1000 microl of DOTMA:DOPE liposomes. Ten patients received VEGF P/L, three patients received beta-galactosidase P/L, and two patients received Ringer lactate. Gene transfer to coronary arteries was feasible and well tolerated. Except for a slight increase in serum C-reative protein in all study groups, no adverse effects or abnormalities in laboratory parameters were detected. No VEGF plasmid or recombinant VEGF protein was present in the systemic circulation after the gene transfer. In control angiography 6 months later, no differences were detected in the degree of coronary stenosis between treatment and control groups. We conclude that catheter-mediated intracoronary gene transfer performed after angioplasty is safe and well tolerated and potentially applicable for the prevention of restenosis and myocardial ischemia.

Background: Periadventitial gene therapy is a promising alternative for the treatment of stenosis, vessel wall thickening and other complications in vascular surgery., Methods: We compared lacZ gene transfer efficiency of DOTMA: DOPE (1:1 w/w) plasmid/liposome complexes and adenoviruses in pig carotid arteries using perivascular delivery with either a collagen collar or a wrap of collagen sheet. Safety of the gene transfer was studied by clinical chemistry, tissue pathology and PCR analysis of lung, liver, kidney, spleen, skeletal muscle and gonads., Results: Gene transfer efficiency using the periadventitial collar was fourfold higher than using the collagen wrap with adenovirus at 7 days (10.22 +/- 2.96 vs 2.78 +/- 1.28 positive cells/mm2; p = 0.18) and 4.3-fold at 14 days (13.46 +/- 3.49 vs 3.11 +/- 0.88 positive cells/mm2; p = 0.03). Gene transfer efficiency at 7 days with adenovirus was fivefold higher than with the plasmid/liposome complexes both using the collar (10.22 +/- 2.96 vs 2.07 +/- 0.95 positive cells/mm2; p = 0.01) and the collagen wrap (2.78 +/- 1.28 vs 0.45 +/- 0.35 positive cells/mm2; p = 0.03). No lacZ activity was detected in plasmid/liposome transfected arteries at 14 days. In spite of the local gene delivery methods a moderate systemic distribution of the transgene was detected in the major organs by PCR analysis., Conclusions: This study shows that: (i) adenovirus delivered with the periadventitial collar or the collagen wrap is well tolerated and may become an efficient new tool in vascular gene therapy, and (ii) gene transfer vector delivered in the periadventitial collar reaches the target tissue more efficiently than the vector in the collagen wrap.

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.

DNA arrays are revolutionizing the analysis of gene expression and single nucleotide polymorphisms of genomic DNA. Currently, the expression of 10-15% of human genes can be analysed simultaneously in a single experiment using cDNA or oligonucleotide-based format of DNA array. Alternatively, smaller DNA arrays with a limited number of selected genes, such as cytokines, growth factors or transcription factors, can be used. In concordance with Human Genome Project, after a few years, the DNA arrays will allow the analysis of expression of the whole human genome and will have a great impact on basic research, drug development and diagnostics. It is important to characterise mechanisms of atherosclerosis-related diseases at the level of gene expression so that new therapeutic strategies can be identified. With the aid of DNA array it is possible to identify multiple, simultaneous, transcriptional events that ameliorate or contribute to atherogenesis. The results are non-physical maps of the function, hierarchy and interactions of genetic programs. In this review we focus on DNA array technology and its applications in atherosclerosis research.

We studied the expression of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), an enzyme capable of hydrolyzing platelet-activating factor (PAF), PAF-like phospholipids, and polar-modified phosphatidylcholines, in human and rabbit atherosclerotic lesions. Oxidative modification of low-density lipoprotein, which plays an important role in atherogenesis, generates biologically active PAF-like modified phospholipid derivatives with polar fatty acid chains. PAF is known to have a potent proinflammatory activity and is inactivated by its hydrolysis. On the other hand, lysophosphatidylcholine and oxidized fatty acids released from oxidized low-density lipoprotein as a result of Lp-PLA(2) activity are thought to be involved in the progression of atherosclerosis. Using combined in situ hybridization and immunocytochemistry, we detected Lp-PLA(2) mRNA and protein in macrophages in both human and rabbit atherosclerotic lesions. Reverse transcriptase-polymerase chain reaction analysis indicated an increased expression of Lp-PLA(2) mRNA in human atherosclerotic lesions. In addition, approximately 6-fold higher Lp-PLA(2) activity was detected in atherosclerotic aortas of Watanabe heritable hyperlipidemic rabbits compared with normal aortas from control rabbits. It is concluded that (1) macrophages in both human and rabbit atherosclerotic lesions express Lp-PLA(2), which could cleave any oxidatively modified phosphatidylcholine present in the lesion area, and (2) modulation of Lp-PLA(2) activity could lead to antiatherogenic effects in the vessel wall.

Liver-directed gene therapy is a promising alternative for the treatment of various liver diseases. Pseudotyped (VSV-G) retroviruses can be produced in high titres which is essential to overcome the problem of low gene transfer efficiency detected previously with first generation Moloney murine (MMLV) retroviruses and plasmid vectors. We compared the lacZ gene transfer efficiency of MMLV retroviruses and VSV-G retroviruses in Watanabe heritable hyperlipidaemic rabbit liver using an intraportal administration route. Hepatocyte proliferation was stimulated by a partial (10%) liver resection and a thymidine kinase-ganciclovir treatment. We also studied the safety of the gene transfer by clinical chemistry, tissue pathology and PCR analysis of lung, kidney, spleen and gonads. Gene transfer efficiency with the VSV-G retrovirus was significantly higher than with the traditional MMLV-based retrovirus (9.5+/-5.26 vs 0.21+/-0.10 positive hepatocytes mm(-2), P<0.05). After a 12-month follow-up period no lacZ expression was detected in liver samples. No transgene was detected in plasma or in lung, kidney, spleen and gonads by PCR analysis 7 days after gene transfer. Transient increases were found in plasma c-reactive protein, aspartyl aminotransferase and alanine aminotransferase levels shortly after the operation with both types of retroviruses. VSV-G retrovirus was well tolerated and may become an efficient new tool in liver gene therapy. The absence of transgene in systemic circulation or in extrahepatic tissues including gonads is an important safety feature required for in vivo gene therapy., (Copyright 1999 Academic Press.)

Extracellular superoxide dismutase (EC-SOD) protects arteries against deleterious effects of superoxide anions and the development of atherosclerosis. In this study, we cloned and characterized rabbit ec-sod gene. We identified 6 rabbit C-elements and 5 CpG clusters in the cloned sequence. One of the CpG clusters is located on the coding sequence. Because CpG clusters are potential sites for methylation and may explain the occurrence of mutations, methylation status of each of the CpG dimers located in the coding sequence CpG cluster was characterized using direct genomic sequencing. Unexpectedly, a marked reduction in the amount of methylated CpG dinucleotides in ec-sod gene was detected in atherosclerotic aortas as compared with normal aortic intima-media. Although alterations in DNA methylation are well characterized in malignant tumors, the presence of methylation changes in atherosclerosis has not been studied even though both diseases are characterized by excess cellular proliferation and alterations in gene expression. Further analysis of the whole genomic methylation by high-pressure liquid chromatography in normal and atherosclerotic aortas revealed a tendency for a decreased 5-methylcytosine (5-mC) content in atherosclerotic aortas as compared with normal arteries. Hypomethylation in atherosclerotic aortas occurred at the same level as has been reported from malignant tumors. Although a causal relationship between the methylation level and expression of EC-SOD cannot be proven, our results show that ec-sod hypomethylation is associated with the development of atherosclerosis and suggest that it may affect structure and function of ec-sod and other genes possibly involved in the development of atherosclerotic lesions.

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.

The class A scavenger receptors (SR-As) are trimeric, integral membrane glycoproteins that exhibit unusually broad ligand-binding properties. A number of studies have suggested that these receptors may play an important role in host defense and in many macrophage-associated pathological processes, including atherosclerosis and Alzheimer's disease. The study of the expression and function of these receptors in human disease has been hampered by the lack of suitable antibodies recognizing human SR-A. This has generated questions regarding the nature of receptors responsible for scavenger receptor activity detected in a variety of cell types, including monocytes, macrophages, smooth muscle cells, and endothelial cells. To address these questions, we have produced high-titer antisera recognizing human SR-A by using mice deficient for SR-A (SR-A -/-). We show that SR-A -/- mice produce a significantly higher-titer immune response than do wild-type (SR-A +/+) littermates, with antisera of the former having a broad species reactivity and recognizing SR-A from humans, mice, and rabbits. The antisera recognize both type I and II SR-A in a wide range of immunological techniques. Using these antisera we show that the expression of SR-A protein is induced during monocyte to macrophage differentiation and that SR-A mediates 80% of the uptake of acetylated low density lipoprotein by human monocyte-derived macrophages. We also establish that human SR-A is expressed by tissue macrophages in liver and lung and by macrophage-derived foam cells within aortic atherosclerotic lesions, with little detectable expression by smooth muscle cells or aortic endothelium.

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.

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.

To study the expression of the bovine beta-lactoglobulin (BLG) gene we isolated the BLG gene from a genomic library and introduced it into murine germline. Bovine BLG gene including 2.8 kbp of 5' and 1.9 kbp of 3' flanking region was expressed efficiently and mammary gland-specifically in transgenic mice. Expression levels of BLG in milk exceeded 1 mg ml-1 in all four mouse lines analyzed. However, in two mouse lines originating from female founders BLG expression levels varied from less than 0.02 mg ml-1 up to 1 mg ml-1. In both lines originating from male founders all analyzed female mice excreted bovine BLG into their milk at a high and constant level of 1-2 mg ml-1. BLG expression was stable within individual mice in two successive lactations and the amount of BLG in the milk of mice correlated with the level of BLG mRNA in the mammary tissue. Methylation analyses of HpaII sites revealed that transgene copies were on average more methylated in mice which excreted low levels of BLG into their milk. Each mouse line had its own methylation pattern and, in addition, each mouse had more or less identical methylation patterns in mammary gland, brain and kidney DNA. Genomic sequencing of the BLG gene indicated that the promoter region (bases -162 to +391 with respect to the transcription start site) was heavily methylated except for distinct CpG sites that were only partially methylated both in transgenic mice and lactating cattle.

Germline mutations of the putative tumor suppressor gene APC are associated in high frequency with the familial adenomatous polyposis, predisposing the patients to colorectal neoplasia. Similarly, sequence analyses have revealed that in more than half of patients with sporadic colorectal carcinoma or adenoma, the APC gene was mutated. By employing genomic sequencing, i.e., base-specific analysis of methylated cytosines, we show here that the promoter region of the APC gene is heavily methylated at CpG sites in patients with colorectal carcinoma in comparison with normal colonic mucosa and premalignant adenomas. Our results suggest that cytosine methylation of the regulatory sequences of the APC gene could be involved in the progression of human colorectal cancer.

DNA methylation has been studied intensively during the past years in order to elucidate its role in the regulation of gene expression, gene imprinting and cancer progression. Earlier studies have shown that a general genomic under-methylation is associated with chronic lymphocytic leukemia and metastatic prostate cancer. Site-specific methylation changes, as revealed by the use of methylation-sensitive restriction enzymes, have been reported to occur in the promotor region of the calcitonin gene in chronic myeloid leukemia as it progresses from the chronic phase to blast crisis, in non-Hodgkin's lymphoid neoplasms and in non-lymphocytic leukemia. We have now explored possible methylation changes associated with benign and malignant breast tumors. Two approaches were employed: (i) chemical determination of general genomic methylation status and (ii) base-specific analysis of the methylation changes in the promoter of the calcitonin gene with the aid of genomic sequencing. The results did not reveal any changes of total DNA 5-methylcytosine content in ductal carcinoma of breast in comparison with benign tumors. There was a small, yet significant, increase in 5-methylcytosine content in lobular carcinoma. Genomic sequencing of the promoter region of the calcitonin gene, however, revealed a striking hypermethylation at or around the transcription start site of the gene in ductal carcinomas. In benign tumors and lobular carcinomas, this region was either entirely unmethylated or only slightly methylated. The latter changes may reflect a regional hypermethylation of the short arm of chromosome 11, which harbors, in addition to the calcitonin gene, a number of putative or established tumor-suppressor genes. Our results demonstrate that genomic sequencing in its present form can be used for a reliable and precise DNA methylation analysis of primary human tumors.