Original Research: Local TAT-p27 Kip1 Fusion protein inhibits cell proliferation in rat Carotid arteries

Abstract

Introduction: p27^Kip1 is a cyclin kinase inhibitor that induces cell cycle arrest. In this study, the efficacy of fusion protein TAT- p27^Kip1 to inhibit cell proliferation in rat perivascular injured carotid arteries was tested.

Methods: The cDNA of p27^Kip1 and GFP (green fluorescein protein) fused to the TAT epitope, which allows cell penetration, yielded TAT-p27 ^Kip1 and TAT-GFP fusion proteins. In vitro biological activity on cell proliferation was evaluated by [³H] thymidine DNA incorporation in rabbit aortic endothelial cells (REC). An in vivo model used a silicone collar filled with saline positioned around the carotid vessel for 14 days to produce an increased adventitia cross-sectional area.

Results: TAT-p27^Kip1 inhibited REC proliferation in vitro using either 100, 200, and 500 nM compared to control (88.2 ± 4.4, 81.3 ± 7, 71.9 ± 4.2 vs. 100 ± 6.7%, N = 3, respectively, p < 0.05). This response was stable for purified proteins stored at -20•C for at least 23 days. In vivo , TAT-p27^Kip1 solution reduced adventitia cross-sectional area in a dose-dependent manner compared to TAT-GFP (area in mm² — TAT-p27^Kip1: 200 nM, 0.160 ± 0.018; 500 nM, 0.050 ± 0.005 vs. TAT-GFP: 500 nM, 0.595 ± 0.066 vs. the contralateral: 0.047 ± 0.005, N = 7, p < 0.01).

Conclusion: Taken together, these results provide evidence that TAT-p27^Kip1 can inhibit vascular cells proliferation. It is the first successful demonstration that the cell permeable TAT-p27^Kip1 has potential as a vascular anti-proliferative agent.