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Release of active and depot GDF-5 after adenovirus-mediated overexpression stimulates rabbit and human intervertebral disc cells

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Abstract

To develop new therapeutic options for the treatment of disc degeneration we tested the possibility of overexpression of active growth and differentiation factor (GDF) 5 and of transforming growth factor (TGF) β1 by adenoviral gene transfer and characterized its effect on cell proliferation and matrix synthesis of cultured rabbit and human intervertebral disc cells. Recombinant adenovirus encoding for GDF-5 or TGF-β1 was developed and transgene expression characterized by RT-PCR, western blot and ELISA. Growth and matrix synthesis of transduced cells was measured by [3H]thymidine or [35S]sulfate incorporation. Disc cells expressed the receptors BMPR1A, BMPR1B, and BMPR2, which are relevant for GDF-5 action. Adenovirus efficiently transferred the GDF-5 gene or the TGF-β1 gene to rabbit and human intervertebral disc cells. About 50 ng GDF-5 protein/106 cells per 24 h or 7 ng TGF-β1 protein/106 cells per 24 h was produced. According to western blotting, two GDF-5 forms, with molecular weights consistent with the activated GDF-5 dimer and the proform, were secreted over the 3 weeks following gene transfer. Overexpressed GDF-5 and TGF-β1 were bioactive and promoted growth of rabbit disc cells in monolayer culture. Our results suggest that ex vivo gene delivery of GDF-5 and TGF-β1 is an attractive approach for the release of mature and pre-GDF-5 in surrounding tissue. This leads us to hope that it will prove possible to improve the treatment of degenerative disc disease by means of ex vivo gene transfer of single or multiple growth factors.

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Abbreviations

BMP :

Bone morphogenetic proteins

CHO :

Chinese hamster ovary

ELISA :

Enzyme-linked immunosorbent assay

FCS :

Fetal calf serum

GDF :

Growth and differentiation factor

GFP :

Green fluorescent protein

IVD :

Intervertebral discs

MOI :

Multiplicity of infection

TGF :

Transforming growth factor

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Acknowledgements

We thank Regina Hess and Michael Rühle for technical assistance, Jens Pohl, Biopharm GmbH, for the donation of GDF-5 cDNA, and Sven Schneider for the statistical analysis. This work was supported by a grant from the research fund of the Stiftung Orthopädische Universitätsklinik Heidelberg.

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Authors and Affiliations

  1. Division of Experimental Orthopaedics, University of Heidelberg, Schlierbacher Landstrasse 200a, 69118, Heidelberg, Germany

    Haili Wang, Markus Kroeber, Carsten Schmid & Wiltrud Richter

  2. Biopharm GmbH, Heidelberg, Germany

    Michael Hanke & Rainer Ries

  3. Cardiology, Benjamin Franklin Hospital, Free University Berlin, Berlin, Germany

    Wolfgang Poller

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  1. Haili Wang
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  2. Markus Kroeber
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Correspondence to Wiltrud Richter.

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Wang, H., Kroeber, M., Hanke, M. et al. Release of active and depot GDF-5 after adenovirus-mediated overexpression stimulates rabbit and human intervertebral disc cells. J Mol Med 82, 126–134 (2004). https://doi.org/10.1007/s00109-003-0507-y

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  • DOI: https://doi.org/10.1007/s00109-003-0507-y

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