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

Expression of Vascular Endothelial Growth Factor and Vascular Endothelial Growth Factor Receptor-2 (KDR/Flk-1) in Ischemic Skeletal Muscle and Its Regeneration

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-1α 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