13. EFFECT OF THE HVEGF TRANSFER ON ENDOGENOUS VEGF MRNA EXPRESSION IN A RAT OSTEOBLAST OR FIBROBLAST CULTURE MODEL

Abstract

Purpose: 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 accelerates bone healing of a rabbit tibia segmental bone defect in-vivo, and increases osteoblast proliferation and mineralization in-vitro. The aim of this project was to examine the effect of exogenous human VEGF (hVEGF) on the endogenous rat VEGF messenger RNA (mRNA) expression in a cell-based gene transfer model.

Method: The osteoblasts were obtained from the rat periosteum. The fibroblasts were obtained from the rat dermal tissue. The cells were then cultured to reach 60% confluence and transfected with hVEGF using Superfect. Four groups were:

1. osteoblast-hVEGF,

2. fibroblast-hVEGF,

3. Osteoblasts alone, and

4. Fibroblasts only.

The cultured cells were harvested at 1, 3 and 7 days after the transfection. The total mRNA was extracted (TRIZOL); both hVEGF and rat VEGF mRNA were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and quantified by VisionWorksLS.

Results: The hVEGF mRNA was detected by RT-PCR from transfected osteoblasts after three days of gene transfection. The hVEGF mRNA expression in transfected fibroblasts increased exponentially at days 1, 3 and 7 after the transfection. We compared the endogenous rat VEGF mRNA expression level of the osteoblasts or fibroblasts that were transfected with hVEGF with the cells without the transfection. The hVEGF transfected osteoblasts had a greater rat VEGF mRNA expression than the non-transfected osteoblasts. Furthermore, when hVEGF was transfected to the rat fibroblasts, the endogenous mRNA expression level measured was also greater than that from the non-transfected fibroblasts. Rat VEGF mRNA expression increased in the first three days of the hVEGF transfection, but the expression level was reduced at Day 7.

Conclusion: These results suggest that cell-based hVEGF gene therapy enhances endogenous rat VEGF mRNA expression in both osteoblasts and fibroblasts.