J Korean Orthop Assoc.  2008 Aug;43(4):479-487. 10.4055/jkoa.2008.43.4.479.

HIF-1alpha and VEGF Expression in Fracture Healing

• Kim JJ
• Shon HC
• Chang JS
• Kim JH
• Lee KS
• Kim SW

• KMID: 1480409
• DOI: http://doi.org/10.4055/jkoa.2008.43.4.479

Abstract

PURPOSE: To elucidate the relation between fracture healing and angiogenesis, we checked expression of Hypoxia-inducible factor (HIF) and Vascular endothelial growth factor (VEGF) in hypoxic cell cultures and the callus from a rat femur fracture model.
MATERIALS AND METHODS
Human osteoblasts, chondrocytes, and rat ST2 cells were cultured in DME/F12 media with 10% FBS. Hypoxic DME/F12 media (PO2<60 mmHg) was generated by bubbling with 95% N2 and 5% CO2 and added to cells. After 2, 6, and 24 hours, RNA and proteins were collected for reverse transcription - polymerase chain reaction (RT-PCR) and Western blot. In addition, immunocytochemistry and siRNA treatment for HIF-1alpha were performed. Next, femurs from 9-week SD rats were fractured after fixation with needles. The rats were sacrificed at post-fracture day (PFD) 3, 5, 7, 10, 14, 21 and calluses were collected for RT-PCR and Western blot.
RESULTS
HIF-1alpha and HIF-2alpha expression were not increased in RT-PCR but protein levels were increased. VEGF expression in RT-PCR was increased. Treatment with siRNA directed towards HIF inhibited VEGF expression. In the rat fracture callus, HIF-1alpha and VEGF expression peaked between PFD 5 and 7 and decreased after PFD 10. In contrast to cell culture, mRNA expression of HIF-1alpha was increased at PFD 7.
CONCLUSION
HIF-1alpha and VEGF peaked early in fracture healing. With expression decreasing as O2 tension increased. Further study is needed to identify other factors affecting chondrogenic differentiation.

Keyword

HIF-1alpha; VEGF; Fracture healing

MeSH Terms

Animals
Blotting, Western
Bony Callus
Cell Culture Techniques
Chondrocytes
Femur
Fracture Healing
Humans
Immunohistochemistry
Needles
Osteoblasts
Polymerase Chain Reaction
Proteins
Rats
Reverse Transcription
RNA
RNA, Messenger
RNA, Small Interfering
Vascular Endothelial Growth Factor A
Proteins
RNA
RNA, Messenger
RNA, Small Interfering
Vascular Endothelial Growth Factor A

Figure

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  Fig. 1 Hypoxia does not induce HIF-1α mRNA expression (A, B) but increases protein levels (C, D). N, normoxia; H, hypoxia.

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  Fig. 2 Hypoxia does not induce HIF-2α mRNA expression (A, B) but increases protein levels (C, D).

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  Fig. 3 Hypoxia increases VEGF mRNA expression in ST2 cells (A) and chondrocytes (B).

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  Fig. 4 Hypoxia does not induce HIF-1α and HIF-2α mRNA expression but increases protein levels. HIF-1α, HIF-2α and VEGF expression in human osteoblasts shows similar expression patterns to ST2 cells and chondrocytes.

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  Fig. 5 Human chondrocyte immunocytochemistry shows increased expression of HIF and VEGF in hypoxia.

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  Fig. 6 VEGF expression was decreased by treatment with siRNA for HIF-1α in human osteoblasts. Mock, transfection control.

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  Fig. 7 VEGF expression was decreased by treatment with siRNA for HIF-1α in human chondrocytes. Mock, transfection control.

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  Fig. 8 Western blot for HIF-1α in callus shows elevation at early stages of fracture healing (peak at PFD 5) and decreases after PFD 10.

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  Fig. 9 RT-PCR for HIF-1α in callus shows elevation at early stages of fracture healing (peak at PFD 7).

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  Fig. 10 RT-PCR for VEGF in callus shows elevation at early stages of fracture healing (peak at PFD 5) and decreases after PFD 10.

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