Abstract 2057: Over-expression of p27 Inhibits Vascular Smooth Muscle Cells Proliferation and Neointima Formation in Rabbit Vein Graft Model

Abstract

Neointima formation caused by vascular smooth muscle cells (VSMC) proliferation is an early event in vascular remodeling associated with arterialization of vein graft and the magnitude of this process may contribute for late failure. Cell cycle is tightly regulated and cyclin-dependent kinase inhibitor p27 kip1 plays a critical role in this process. We investigated whether overexpression of p27 can modulate this early cell proliferative response in rabbit vein grafts. First generation bicistronic adenovirus AdCMVp27hEGFP, (AV1) harbouring the human p27 sequence, and AdCMVLacz (AV2) were engineered. Overexpression of p27 was analyzed by immunofluorescence and the antiproliferative effect of AV1 was assessed in primary culture of VSMC followed by 1, 3, 5, and 9 days after AV1 transduction vs. AV2. A segment of jugular vein vas exposed to AV1 and AV2 (10 9 –10 11 pfu/mL) or placebo for 30 min and then interposed, end to end, to the carotid artery of adult male New Zealand rabbits. After 28 days the vein grafts were harvested and stained with Miller for neointima analyses. Imunofluorescence of VSMC showed positive cells 72 hs following AV1 transduction. Significant inhibition of cell proliferation was observed 7 and 9 days after the cells were exposed to AV1 compared to AV2 or control cells (7 days: 0.34 ± 0.15 × 10 5 vs. 1.5 ± 0.22 × 10 5 , 2.04 ± 0.29 × 10 5 cell/ml, p<0.01; 9 days: 0.23 ± 0.1 × 10 5 vs. 1.49 ± 0.31 × 10 5 , 2.31 ± 0.41 × 10 5 cell/ml, p<0.01). In vivo , transduction efficiency was evaluated by the expression of B-galactosidase 48 hs. after surgery in vein grafts exposed to AV2. Vein grafts transduced with AV1 showed significant reduction in neointima hyperplasia compared with AV2 or to control animals not exposed to any viral treatment (47.33 ± 5.36 vs. 112.83 ± 24.2 and 80.08 ± 10.51μm, p<0.05). These results show that adenovirus mediated over expression of human protein p27 can inhibit VMSC proliferation in vitro and significantly impair the neointima formation associated with arterialization of vein graft after 28 days. It will important to establish the long term effects of this intervention specially when associated with other environment stimuli such as high fat that can clearly influence the outcome of this therapeutic intervention.