[Effects of myocardial transplantation of mesenchymal stem cells transfected with vascular endothelial factor gene on improvement of heart function and angiogenesis after myocardial infarction: experiment with rats].
To establish a method to transfect vascular endothelial factor (VEGF) gene into mesenchymal stem cells ( MSCs) , to investigate the effects of the gene-transfected MSCs on heart function restoration and angiogenesis after myocardial infarction, and to compare the differences among cell therapy, gene therapy, and combined therapy.


METHODS
Seventy-one Wistar rats underwent ligation of the left anterior descending coronary artery so as to establish heart ischemia models. Fifteen rats underwent sham operation. MSCs were isolated from several Wistar rats by density gradient centrifugation, purified, and transfected with pcDNA3.1-hVEGF165 or blank plasmid pcDNA3.1 respectively using the liposome mediated method. ELISA, Western blotting, and RT-PCR were used to detect the protein and mRNA expression of hVEG in these MSCs Forty-eight surviving rats that underwent ligation were randomly divided into 4 equal groups: combination group (Combo group) to be injected into the heart infarct zone with suspension of hVEGF165-transfected MSCs 2 weeks after the establishment of the model, cell group to be injected with suspension of MSCs not transfected with VEGF, gene group to be injected with suspension of DNA-liposome containing pcDNA3.1-VEGF165 and control group to be injected with cold culture fluid only. Twelve surviving rats that underwent sham operation were used as normal non-ischemic group. Four weeks after the injection the surviving rats underwent examination of heart functions by the Buxco system. The rats were killed and their hearts were taken out to undergo immunohistochemistry with 5-bromodeoxyuridine (Brdu) and troponin T and factor VIII to measure the area of cardiac infarction and the capillary density. RT-PCR was used to examine the mRNA expression of VEGF. The heart infarcted size was calculated by Evan's blue staining.


RESULTS
(1) MSCs can be successfully isolated and cultured by density gradient centrifugation followed by adherence-separation. The expression of hVEGF165 in the transfected MSCs was demonstrated with ELISA, RT-PCR and Western Blot Assay. (2) Four weeks after the cells were transplanted, among all groups but the nonischemic group, the heart infarcted size of the Combo group was 27.8% +/- 3. 0% ,significantly less than those of the cell group (37.0% +/- 10. 1% ) and gene group (37.1% +/- 5.2%, both P <0.05). The heart function of the Combo group was better than those of other groups. (3) The capillary density of the Combo group was 40. 2 +/- 5.5/visual field, significantly greater than those of both the cell group (27.2 +/- 6. 3/visual field, P <0. 01) and that of the control group (18.5 +/- 5.8/visual field, P <0. 01) , and greater to some degree than that of the gene group (35. 8 +/-7.7/visual field, P =0. 189). (4)The heart infarcted size, heart function and capillary density of the cell and gene groups were similar and were smaller, better and greater than those of the control group. (5) Brdu and troponin T double staining detected a varied increase in the number of surviving cardiomyocytes at the heart infarcted area, some of which were double stain positive. RT-PCR showed mRNA expression of hVEGF165 in the Combo and gene groups, that in the Combo group being higher than that in the gene group.


CONCLUSIONS
Eukaryotic expression vector pcDNA3.1-hVEGF165 can effectively be expressed in MSCs. Transplantation of VEGF gene by means of transfected MSCs brings better improvement in myocardial perfusion and in restoration of heart function than either cellular or gene therapy alone.
Yang
Jin-fu
JF
Department of Cardiothoracic Surgery, Second Xiangya Hospital, Central South University, Changsha 410011, China.
Zhou
Wen-wu
WW
Tang
Tao
T
Yu
Jie-feng
JF
Zhou
Xin-min
XM
Hu
Jian-guo
JG
D000818:Animals; D015153:Blotting, Western; D015316:Genetic Therapy; D006321:Heart / Q000503:physiopathology; D066298:In Vitro Techniques; D008297:Male; D045164:Mesenchymal Stem Cell Transplantation; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D009203:Myocardial Infarction / Q000473:pathology / Q000503:physiopathology / Q000601:surgery*; D009206:Myocardium / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000502:physiology; D011897:Random Allocation; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism
C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A