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

Heart Institute (InCor)/LIM 13, University of Sao Paulo Medical School, Sao Paulo, Brazil

Ayumi A. Miyakawa

Laboratory of Genetic and Molecular Cardiology, Heart Institute (InCor), University of Sao Paulo Medical School, Av. Dr. Eneas C. Aguiar, 44 - 10 andar, 05403-000 Sao Paulo - SP - Brazil, ayumi.miyakawa{at}incor.usp.br

Sandra Y. Fukada

Department of Pharmacology, School of Medicine of Ribeirao Preto, USP, Ribeirao Preto SP, Brazil

Claudia R. de Andrade

Department of Pharmacology, School of Medicine of Ribeirao Preto, USP, Ribeirao Preto SP, Brazil

Fernando P. Pacheco

Heart Institute (InCor)/LIM 13, University of Sao Paulo Medical School, Sao Paulo, Brazil

Thaís G. da Silva

Heart Institute (InCor)/LIM 13, University of Sao Paulo Medical School, Sao Paulo, Brazil

Leandra N. Z. Ramalho

Department of Pathology, School of Medicine of Ribeirao Preto, USP, Ribeirao Preto SP, Brazil

Ana Maria de Oliveira

Department of Pharmacology, School of Medicine of Ribeirao Preto, USP, Ribeirao Preto SP, Brazil

Jose E. Krieger

Laboratory of Genetic and Molecular Cardiology, Heart Institute (InCor), University of Sao Paulo Medical School, Av. Dr. Eneas C. Aguiar, 44 - 10 andar, 05403-000 Sao Paulo - SP - Brazil, krieger{at}incor.usp.br

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.

Key Words: p27Kip1 • TAT-fusion protein • vascular injury

Therapeutic Advances in Cardiovascular Disease, Vol. 2, No. 3, 129-136 (2008)
DOI: 10.1177/1753944708090170


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