[Local shRNA modulation of phosphatidylinositol 3-kinase signaling pathway reduces intimal hyperplasia in vein grafts: experiment with rats].
OBJECTIVE
To investigate whether knockdown Pik3cb p110beta subunit by shRNA in autologous vein grafts can reduce intimal hyperplasia.


METHODS
180 adult SD rats underwent carotid artery bypass graft surgery by using the autologous branch of jugular vein, and they were randomly divided into 6 equal groups: Group A (with the jugular vein grafts treated with 25% Pluronic F-127 only), Group B (with the graft treated with the plasmid encoding shRNA targeting Pik3cb p110beta subunit, pU6-Pik3cb-shRNA-1), Group C (with the graft treated with the plasmid encoding shRNA targeting Pik3cb p110beta subunit, pU6-Pik3cb-shRNA-2), Group D (with the graft treated with the half pU6-Pik3cb-shRNA-1 and pU6-Pik3cb-shRNA-2), and Group E (with the graft treated with the pGenesil-1 scramble shRNA), and Group F (with the jugular vein grafts treated with wortmannin). Specimens of jugular vein graft were harvested 1, 3, 7, 14, and 28 days after surgery to assess the neointimal hyperplasia. Another 18 rats were randomly divided into 6 equal groups as mentioned above to be used in a parallel experiment: 72 h after surgery specimens of jugular vein graft were harvested to undergo fluorescence quantitative real-time PCR and Western blotting to detect the mRNA expression of P13K p110beta subunit and the protein expression of phosphorylated Akt - phospho-Akt (Thr 308) and phospho-Akt (Ser473)-, and mTOR (Ser 2448). And another 9 rats received jugular vein grafts treated with the pGenesil-1 scramble shRNA, and on the postoperative days 1, 2, and 3 respectively 3 rats were killed to undergo fluorescence staining to detect the transfection efficacy.


RESULTS
The transfection rate of the plasmid pGenesil-1 was 60% in the vascular smooth muscle cells and 90% in the endothelial cells. The thickness of tunica intima 28 days after the surgery of the pU6-Pik3cb-shRNA-1, pU6-Pik3cb-shRNA-2, 1/2 (shRNA1 + shRNA2), and wortmannin groups were (34.6 +/- 2.7) microm, (39.4 +/- 2.5) microm, (36.7 +/- 2.9) microm, and (40.6 +/- 3.1) microm respectively, all significantly lower than that of the control group (61.8 +/- 4.3 microm, P < 0.05). The expression levels of phospho-Akt (Thr 308), phospho-Akt (Ser 473), and mTOR of the shRNA intervention and wortmannin groups were all down-regulated.


CONCLUSIONS
Knockdown of Pik3cb in interposition carotid artery branch of jugular vein grafts reduces intimal hyperplasia with the possible mechanism of downregulation of phosphatidylinositol 3-kinase signaling through Akt, with resultant decreases in VASC growth and survival. Modulation of the phosphatidylinositol 3-kinase pathway through knockdown Pik3cb may represent a novel therapy to prevent vein graft intimal hyperplasia after bypass grafting.
Deng
Yong-zhi
YZ
Department of Cardiovascular Surgery, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Liu
Su-jian
SJ
Ma
Li
L
Sun
Zong-quan
ZQ
Chen
Jia-jun
JJ
Su
Gang
G
Liu
Chao
C
Wang
Guo-hua
GH
Ke
Jun
J
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002339:Carotid Arteries / Q000601:surgery; D004195:Disease Models, Animal; D006083:Graft Occlusion, Vascular / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006965:Hyperplasia; D007601:Jugular Veins / Q000637:transplantation; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D011897:Random Allocation; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D017539:Tunica Intima / Q000378:metabolism / Q000473:pathology; D000077191:Wortmannin