In vitro functional assessment of human skeletal myoblasts after transduction with adenoviral bicistronic vector carrying human VEGF165 and angiopoietin-1.
OBJECTIVE
We report in vitro functional assessment of human skeletal myoblasts with adenoviral bicistronic vector carrying human vascular endothelial growth factor-165 (hVEGF165) and angiopoietin-1 (Ang-1).


METHODS
Myoblasts were assessed for their purity by desmin expression. A replication incompetent adenoviral bicistronic vector (Ad-Bic) carrying both hVEGF165 and Ang-1 was used for transduction of myoblasts. Transduction efficiency was assessed by dual fluorescent immunostaining of the transduced myoblasts. Expression efficiency was analyzed by enzyme linked immunosorbent assay (ELISA), Western blot and reverse transcription polymerase chain reaction (RT-PCR). The biological activity of the secreted human VEGF165 and Ang-1 was determined by human umbilical vein endothelial cells (HUVEC) proliferation assay, Thymidine [H3] incorporation assay and capillary-like structure formation.


RESULTS
The myoblasts preparation was >98% pure. Fluorescent immunostaining showed >95% transduction efficiency. The transduced myoblasts secreted VEGF(165) for up to 30 days after transduction, with peak level (32 +/- 4 ng/ml) at day 8 after transduction as revealed by VEGF ELISA. Western blot further confirmed that both angiogenic factors were actively secreted by transduced myoblasts. The molecular weight was 42 kD for hVEGF165 and 70 kD for Ang-1 respectively. The expression of hVEGF165 and Ang-1 was significantly reduced at day-30 after transduction as seen by RT-PCR. The conditioned medium from bicistronic vector transduced myoblasts stimulated HUVEC to proliferate much faster than other conditioned media (>1.5 folds). Thymidine incorporation assay further confirmed this finding. Matrigel experiment suggested that HUVEC under the condition of both growth factors formed significantly more capillary-like structure.


CONCLUSIONS
The bicistronic vector transduced myoblasts provides a novel strategy for therapeutic angiomyogenesis for cardiac repair.
Ye
Lei
L
Department of Cardiothoracic and Vascular Surgery, National University of Singapore, Singapore.
Haider
Husnain Kh
HKh
Jiang
Shujia
S
Ge
Ruowen
R
Law
Peter K
PK
Sim
Eugene K W
EK
D000256:Adenoviridae / Q000235:genetics; D042683:Angiopoietin-1 / Q000235:genetics* / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D032448:Myoblasts, Skeletal / Q000378:metabolism*; D018919:Neovascularization, Physiologic / Q000502:physiology; D014161:Transduction, Genetic; D019076:Transgenes / Q000502:physiology; D014471:Umbilical Veins; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism
D042683:Angiopoietin-1; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A