Effect of human vascular endothelial growth factor gene transfer on endogenous vascular endothelial growth factor mRNA expression in a rat fibroblast and osteoblast culture model.
OBJECTIVE
Vascular endothelial growth factor (VEGF) plays an important role in promoting angiogenesis and osteogenesis during fracture repair. Our previous studies have shown that cell-based VEGF gene therapy enhances bone healing of a rabbit tibia segmental bone defect in vivo. The aim of this project was to examine the effect of exogenous human VEGF on the endogenous rat VEGF messenger RNA (mRNA) expression in a cell-based gene transfer model.


METHODS
Rat fibroblasts and osteoblasts were harvested from the dermal tissue and periosteum, respectively, of Fisher 344 rats. The cells were then cultured and transfected with pcDNA-human VEGF using Superfect reagent (Qiagen). Four experimental groups were created: 1) fibroblast-VEGF; 2) osteoblast-VEGF; 3) nontransfected fibroblast controls; and 4) nontransfected osteoblast controls. The cultured cells were harvested at 1, 3, and 7 days after the gene transfection. The total mRNA was extracted (Trizol; Invitrogen); both human VEGF and rat VEGF mRNA were measured by reverse transcriptase-polymerase chain reaction and quantified by VisionWorksLS.


RESULTS
The human VEGF165 mRNA was detected by reverse transcriptase-polymerase chain reaction from transfected fibroblasts and osteoblasts at 1, 3, and 7 days after gene transfection. The human VEGF165 levels peaked at Day 1 and then gradually reduced expression in both transfected fibroblasts and osteoblasts. Two endogenous rat VEGF isoforms were detected in this cell culture model: rat VEGF120 and rat VEGF164. We compared the rat VEGF120 and rat VEGF164 expression level of the fibroblasts or osteoblasts that were transfected with human VEGF165, with nontransfected control cells. Both the transfected fibroblasts and osteoblasts showed greater expression of rat VEGF164 than nontransfected controls at Day 1 (peak level) and Day 3, but not at Day 7. The expression of rat VEGF120 was lower in transfected fibroblasts, but higher in transfected osteoblasts, than the relevant control groups at any time point after transfection. In addition, human VEGF gene transfection increased osteoblast cell proliferation after 3 days.


CONCLUSIONS
These in vitro results suggest that cell-based human VEGF gene therapy is not only effective at causing human VEGF expression, but also enhances endogenous rat VEGF mRNA expression in both fibroblasts and osteoblasts, particularly the rat VEGF164 isoform.
Li
Ru
R
Musculoskeletal Research Laboratory, St. Michael's Hospital, Division of Orthopaedic Surgery, University of Toronto, Toronto, Ontario, Canada. lir@smh.toronto.on.ca
Li
Claire H
CH
Nauth
Aaron
A
McKee
Michael D
MD
Schemitsch
Emil H
EH
D000818:Animals; D002452:Cell Count; D049109:Cell Proliferation; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D020033:Protein Isoforms; D011817:Rabbits; D051381:Rats; D011916:Rats, Inbred F344; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism
D020033:Protein Isoforms; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A