Local TAT-p27Kip1 fusion protein inhibits cell proliferation in rat carotid arteries.
BACKGROUND
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 [(3)H] 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 mm(2) - 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).


CONCLUSIONS
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.
Neukamm
Birgit
B
Heart Institute (InCor)/LIM 13, University of Sao Paulo Medical School, Sao Paulo, Brazil.
Miyakawa
Ayumi A
AA
Fukada
Sandra Y
SY
de Andrade
Claudia R
CR
Pacheco
Fernando P
FP
da Silva
Thaís G
TG
Ramalho
Leandra N Z
LN
de Oliveira
Ana Maria
AM
Krieger
Jose E
JE
D000818:Animals; D001011:Aorta / Q000166:cytology; D002339:Carotid Arteries / Q000166:cytology*; D020212:Carotid Artery Injuries / Q000473:pathology; D049109:Cell Proliferation; D002478:Cells, Cultured; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000502:physiology*; D042783:Endothelial Cells / Q000166:cytology; D004730:Endothelium, Vascular / Q000166:cytology; D000939:Epitopes; D015696:Gene Products, tat; D008297:Male; D051381:Rats; D017208:Rats, Wistar
D000939:Epitopes; D015696:Gene Products, tat; D050760:Cyclin-Dependent Kinase Inhibitor p27