Release of active and depot GDF-5 after adenovirus-mediated overexpression stimulates rabbit and human intervertebral disc cells |
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Authors: | Haili?Wang Markus?Kroeber Michael?Hanke Rainer?Ries Carsten?Schmid Wolfgang?Poller Email author" target="_blank">Wiltrud?RichterEmail author |
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Institution: | (1) Division of Experimental Orthopaedics, University of Heidelberg, Schlierbacher Landstrasse 200a, 69118 Heidelberg, Germany;(2) Biopharm GmbH, Heidelberg, Germany;(3) Cardiology, Benjamin Franklin Hospital, Free University Berlin, Berlin, Germany |
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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) 1 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- 1 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- 1 gene to rabbit and human intervertebral disc cells. About 50 ng GDF-5 protein/106 cells per 24 h or 7 ng TGF- 1 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- 1 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- 1 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.Abbreviations
BMP
Bone morphogenetic proteins
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CHO
Chinese hamster ovary
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ELISA
Enzyme-linked immunosorbent assay
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FCS
Fetal calf serum
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GDF
Growth and differentiation factor
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GFP
Green fluorescent protein
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IVD
Intervertebral discs
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MOI
Multiplicity of infection
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TGF
Transforming growth factor |
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Keywords: | Gene therapy Intervertebral disc Growth factors Recombinant adenoviral vectors Extracellular matrix |
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