Open Access, Peer-reviewed
pISSN 1226-2102
eISSN 2005-8918
    J Korean Orthop Assoc. 2008 Aug;43(4):479-487. Korean.
    Published online Aug 31, 2008.
    https://doi.org/10.4055/jkoa.2008.43.4.479
    Copyright © 2008 The Korean Orthopaedic Association
    Original Article

    HIF-1α and VEGF Expression in Fracture Healing

    Jung-Jae Kim, M.D., Hyun-Chul Shon, M.D.,* Jae-Suk Chang, M.D., Jung-Hwa Kim, Kang-Sik Lee and Seok-Won Kim, M.D.*
      • Department of Orthopedic Surgery, Asan Medical Center, Ulsan University College of Medicine, Seoul, Korea.
      • *Department of Orthopedic Surgery, Chungbuk National University College of Medicine, Cheongju, Korea.
    • Address reprint requests to Hyun-Chul Shon, M.D. Department of Orthopedic Surgery, Chungbuk National University College of Medicine, 62, Gaeshin-dong, Heungdeock-gu, Cheong-Ju, Chungbuk 360-711, Korea. Tel: +82.43-269-6077, Fax: +82.43-274-8719, Email: hyunchuls@chungbuk.ac.kr

    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-1α 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-1α and HIF-2α 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-1α and VEGF expression peaked between PFD 5 and 7 and decreased after PFD 10. In contrast to cell culture, mRNA expression of HIF-1α was increased at PFD 7.

    Conclusion

    HIF-1α 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.

    Keywords
    HIF-1α; VEGF; Fracture healing

    Figures

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

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

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

    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.

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

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

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

    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.

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

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