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TAT‐p27 fusion protein prevents perivascular injury in rat carotid arteries
Author(s) -
Miyakawa Ayumi Aurea,
Neukamm Birgit,
Fukada Sandra,
Andrade Claudia R,
Pacheco Fernando P,
Ramalho Leandra N. Z.,
Oliveira Ana Maria,
Krieger Jose Eduardo
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a206-a
Subject(s) - green fluorescent protein , carotid arteries , saline , chemistry , fusion protein , in vivo , microbiology and biotechnology , andrology , in vitro , biology , medicine , gene , biochemistry , recombinant dna
The efficacy of p27 protein to inhibit cell proliferation in rat perivascular injured carotid arteries was tested. The cDNA of p27 and GFP (green fluorescein protein) were fused to the TAT epitope, which allows cell penetration, to produce TAT‐p27 and TAT‐GFP fusion proteins. TAT‐p27 showed a dose‐dependent inhibition on cell proliferation in vitro using rabbit aortic endothelial cells (REC) (TAT‐p27 in nM – 0: 100 ± 6.71 %, 100: 88.21 ± 4.45 %, 200: 81.32 ± 6.98 %, 500: 71.92 ± 4.17 %, N = 3, p<0.05). It was stable up to 23 days after purification (TAT‐p27 500nM: no protein: 100 ± 8.53%, days after purification ‐ 2: 71.92 ± 8.25 %, 9: 65.83 ± 13.54 %, 16: 75.77 ± 8.44 %, 23: 75.79 ± 5.40 %, N= 3, p<0.05). The TAT‐GFP used as a control resulted in no alteration in [ 3 H] timidine uptake. For the in vivo experiment, a silicone collar filled with saline was positioned around the rat right common carotid artery for 14 days, which is associated to increased adventicia cross‐sectional area compared to the contralateral arteries while no modification in intimal or medial layers are observed. TAT‐p27 added to the saline solution reduced adventicia cross‐sectional area in a dose‐dependent manner in contrast to TAT‐GFP (area in mm 2 ‐ 500nM TAT‐GFP: 0.595 ± 0.066, 100nM TAT‐p27: 0.160 ± 0.018, 500nM TAT‐p27: 0.050 ± 0.005; contralateral: 0.047 ± 0.005, N=7, p<0.01). The results demonstrate that TAT‐p27 inhibits cell proliferation in vitro and in vivo . The purified TAT‐p27 maintains biological activity up to 23 days and its potential therapeutic applications in vascular injury shall be explored.