Extracellular superoxide dismutase with vaccinia virus anti-inflammatory protein 35K or tissue inhibitor of metalloproteinase-1: Combination gene therapy in the treatment of vein graft stenosis in rabbits.

Bypass graft surgery is limited by stenosis of vein grafts. Neointimal formation in vein graft stenosis is affected by oxidative stress, acute inflammatory response, and proliferation. Gene therapy offers a novel treatment strategy for vein graft stenosis because gene transfer can be done ex vivo during the graft operation. In this study we used adenovirus-mediated ex vivo gene transfer of extracellular superoxide dismutase (EC-SOD) alone or in combination with tissue inhibitor of metalloproteinase-1 (TIMP-1) or vaccinia virus antiinflammatory protein 35K to prevent vein graft stenosis in a jugular vein graft model in normocholesterolemic New Zealand White rabbits. Vein grafts were analyzed 14 and 28 days after the gene transfer, using histological methods. It was found that at the 2-week time point EC-SOD + 35K and EC-SOD + TIMP-1 combinations delivered by gene transfer were the most efficient treatments in decreasing neointimal formation. At the 4-week time point the effect was seen only in the EC-SOD + TIMP-1 combination group. The combination of antiinflammatory proteins (EC-SOD + 35K) was the most effective in reducing macrophage accumulation, which was still significant at the 4-week time point, but this did not prevent vein graft thickening. In conclusion, oxidative, inflammatory, and proliferative processes are important for neointimal formation in vein graft stenosis. In the rabbit model of vein graft disease, combination gene therapy with antioxidative, antiinflammatory, and antiproliferative genes was effective in decreasing neointimal formation. This may be because two different genes may more efficiently affect different pathogenetic pathways at the early stage of the disease process than gene transfer approaches based on single genes.

Effects of vaccinia virus anti-inflammatory protein 35K and TIMP-1 gene transfers on vein graft stenosis in rabbits.

BACKGROUND: Vein graft stenosis is a common problem after bypass surgery. Vein grafts are ideal targets for gene therapy because transduction can be made ex vivo before grafting. Since chemokines and inflammatory factors are involved in vein graft thickening, we tested a hypothesis that the vaccinia virus anti-inflammatory protein 35K which can sequester CC-chemokines, can reduce vein graft thickening in vivo. MATERIALS AND METHODS: We used adenovirus-mediated gene transfer (1x10(9) pfu/ml) of 35K and compared its effects on reducing stenosis in a rabbit jugular vein graft model with tissue inhibitor of metalloproteinase-1 (TIMP-1) and LacZ control gene. TIMP-1 was used in this study because it has previously been shown to inhibit vein graft stenosis in other model systems. The expression of transgenes in the transduced segments was confirmed by RT-PCR. Vein grafts were analyzed using immunohistological and morphometric methods at the three-day time-point and at two-week and four-week time-points. RESULTS: It was found that the anti-inflammatory protein 35K was an efficient factor in reducing neointima formation at the two-week time-point, indicating that inflammatory factors play an important role in vein graft stenosis. At the four-week time-point, 35K still showed a reduced accumulation of macrophages. TIMP-1 also tended to reduce neointimal thickening at the two-week time-point as compared to LacZ. CONCLUSION: It was found that 35K is an efficient factor in reducing neointima formation, macrophage accumulation and proliferation in rabbit vein grafts after adenoviral ex vivo gene transfer.

Tissue inhibitor of metalloproteinase 1 adenoviral gene therapy alone is equally effective in reducing restenosis as combination gene therapy in a rabbit restenosis model.

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.

Peptide-retargeted adenovirus encoding a tissue inhibitor of metalloproteinase-1 decreases restenosis after intravascular gene transfer.

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.