Preventing intimal thickening of vein grafts in vein artery bypass using STAT-3 siRNA.
BACKGROUND
Proliferation and migration of vascular smooth muscle cells (VSMCs) play a key role in neointimal formation which leads to restenosis of vein graft in venous bypass. STAT-3 is a transcription factor associated with cell proliferation. We hypothesized that silencing of STAT-3 by siRNA will inhibit proliferation of VSMCs and attenuate intimal thickening.


METHODS
Rat VSMCs were isolated and cultured in vitro by applying tissue piece inoculation methods. VSMCs were transfected with STAT 3 siRNA using lipofectamine 2000. In vitro proliferation of VSMC was quantified by the MTT assay, while in vivo assessment was performed in a venous transplantation model. In vivo delivery of STAT-3 siRNA plasmid or scramble plasmid was performed by admixing with liposomes 2000 and transfected into the vein graft by bioprotein gel applied onto the adventitia. Rat jugular vein-carotid artery bypass was performed. On day 3 and7 after grafting, the vein grafts were extracted, and analyzed morphologically by haematoxylin eosin (H&E), and assessed by immunohistochemistry for expression of Ki-67 and proliferating cell nuclear antigen (PCNA). Western-blot and reverse transcriptase polymerase chain reaction (RT-PCR) were used to detect the protein and mRNA expression in vivo and in vitro. Cell apoptosis in vein grafts was detected by TUNEL assay.


RESULTS
MTT assay shows that the proliferation of VSMCs in the STAT-3 siRNA treated group was inhibited. On day 7 after operation, a reduced number of Ki-67 and PCNA positive cells were observed in the neointima of the vein graft in the STAT-3 siRNA treated group as compared to the scramble control. The PCNA index in the control group (31.3 ± 4.7) was higher than that in the STAT-3 siRNA treated group (23.3 ± 2.8) (P < 0.05) on 7d. The neointima in the experimental group(0.45 ± 0.04 μm) was thinner than that in the control group(0.86 ± 0.05 μm) (P < 0.05).Compared with the control group, the protein and mRNA levels in the experimental group in vivo and in vitro decreased significantly. Down regulation of STAT-3 with siRNA resulted in a reduced expression of Bcl-2 and cyclin D1. However, apoptotic cells were not obviously found in all grafts on day 3 and 7 post surgery.


CONCLUSIONS
The STAT-3 siRNA can inhibit the proliferation of VSMCs in vivo and in vitro and attenuate neointimal formation.
Department of Cardiovascular Surgery, The Second Hospital, Jilin University, Chang Chun, China.
Jiangbin
J
Sun
Jinhua
J
Zheng
Kaitelynne H
KH
Ling
Keyan
K
Zhao
Zhongshang
Z
Xie
Bo
B
Li
Tiance
T
Wang
Zhicheng
Z
Zhu
Amit N
AN
Patel
Weiping
W
Min
Kexiang
K
Liu
Xiufen
X
Zheng
D000818:Animals; D001807:Blood Vessel Prosthesis / Q000009:adverse effects*; D049109:Cell Proliferation; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation / Q000235:genetics; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D019394:Ki-67 Antigen / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000473:pathology; D032389:Myocytes, Smooth Muscle / Q000378:metabolism / Q000473:pathology; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D051381:Rats; D050796:STAT3 Transcription Factor / Q000378:metabolism*; D017539:Tunica Intima / Q000378:metabolism / Q000473:pathology*; D014656:Vascular Surgical Procedures / Q000379:methods*
D019394:Ki-67 Antigen; D018809:Proliferating Cell Nuclear Antigen; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; D050796:STAT3 Transcription Factor; C494088:Stat3 protein, rat; D019938:Cyclin D1