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

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.

[1]  S. Kokubun,et al.  Changes with Age in Proteoglycan Synthesis in Cells Cultured In Vitro From the Inner and Outer Rabbit Annulus Fibrosus: Responses to Interleukin-1 and Interleukin-1 Receptor Antagonist Protein , 2000, Spine.

[2]  M. Sporn,et al.  Physiological Actions and Clinical Applications of Transforming Growth Factor-β (TGF-β) , 1993 .

[3]  M. Sporn,et al.  Transforming growth factor-beta: recent progress and new challenges , 1992, The Journal of cell biology.

[4]  J. Bonaventure,et al.  Effect of transforming growth factor-β1 (TGF-β1) on matrix synthesis by monolayer cultures of rabbit articular chondrocytes during the dedifferentiation process☆ , 1992 .

[5]  J. Adler,et al.  Early Onset of Disk Degeneration and Spondylosis in Sand Rats (Psammomys Obesus) , 1983, Veterinary pathology.

[6]  A. Nerlich,et al.  New In Vivo Animal Model to Create Intervertebral Disc Degeneration and to Investigate the Effects of Therapeutic Strategies to Stimulate Disc Regeneration , 2002, Spine.

[7]  W. Richter,et al.  Molecular analysis of expansion, differentiation, and growth factor treatment of human chondrocytes identifies differentiation markers and growth-related genes. , 2002, Biochemical and biophysical research communications.

[8]  U. Walter,et al.  Endogenous or overexpressed cGMP-dependent protein kinases inhibit cAMP-dependent renin release from rat isolated perfused kidney, microdissected glomeruli, and isolated juxtaglomerular cells. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[9]  S. Lohmann,et al.  cGMP Stimulation of Cystic Fibrosis Transmembrane Conductance Regulator Cl− Channels Co-expressed with cGMP-dependent Protein Kinase Type II but Not Type Iβ* , 1997, The Journal of Biological Chemistry.

[10]  M. Sporn,et al.  Physiological actions and clinical applications of transforming growth factor-beta (TGF-beta). , 1993, Growth factors.

[11]  T. Oegema,et al.  Biochemistry of the intervertebral disc. , 1993, Clinics in sports medicine.

[12]  C HIRSCH,et al.  Studies on structural changes in the lumbar annulus fibrosus. , 1952, Acta orthopaedica Scandinavica.

[13]  V. Rosen,et al.  Recombinant human bone morphogenetic protein induces bone formation. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[14]  N. Boos,et al.  2002 SSE Award Competition in Basic Science: Expression of major matrix metalloproteinases is associated with intervertebral disc degradation and resorption , 2002, European Spine Journal.

[15]  B. Vernon‐roberts,et al.  A longitudinal study of the matrix changes induced in the intervertebral disc by surgical damage to the annulus fibrosus , 1992, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[16]  H E Gruber,et al.  Human intervertebral disc cells from the annulus: three-dimensional culture in agarose or alginate and responsiveness to TGF-beta1. , 1997, Experimental cell research.

[17]  J. McPherson,et al.  Synergistic action of transforming growth factor-beta and insulin-like growth factor-I induces expression of type II collagen and aggrecan genes in adult human articular chondrocytes. , 1997, Experimental cell research.

[18]  F. Luyten,et al.  Cartilage-derived morphogenetic proteins , 1997 .

[19]  J A Buckwalter,et al.  Aging and degeneration of the human intervertebral disc. , 1995, Spine.

[20]  J. Adler,et al.  Degeneration of the intervertebral disks and spondylosis in aging sand rats. , 1979, Archives of pathology & laboratory medicine.

[21]  James D. Kang,et al.  Human Intervertebral Disc Cells Are Genetically Modifiable by Adenovirus-Mediated Gene Transfer: Implications for the Clinical Management of Intervertebral Disc Disorders , 2000, Spine.

[22]  J. Bonaventure,et al.  Effect of transforming growth factor-beta 1 (TGF-beta 1) on matrix synthesis by monolayer cultures of rabbit articular chondrocytes during the dedifferentiation process. , 1992, Experimental cell research.

[23]  M. Ferguson,et al.  Transforming growth factor beta s and wound healing. , 1997, The international journal of biochemistry & cell biology.

[24]  L G Gilbertson,et al.  Modulation of the biologic activity of the rabbit intervertebral disc by gene therapy: an in vivo study of adenovirus-mediated transfer of the human transforming growth factor beta 1 encoding gene. , 1999, Spine.

[25]  F. Graham,et al.  Manipulation of adenovirus vectors. , 1991, Methods in molecular biology.

[26]  S. Lohmann,et al.  Endogenous Type II cGMP-dependent Protein Kinase Exists as a Dimer in Membranes and Can Be Functionally Distinguished from the Type I Isoforms* , 1997, The Journal of Biological Chemistry.

[27]  T. Oegema,et al.  Identification of heterogeneous cell populations in normal human intervertebral disc. , 1995, Journal of anatomy.

[28]  M. Heberer,et al.  Specific growth factors during the expansion and redifferentiation of adult human articular chondrocytes enhance chondrogenesis and cartilaginous tissue formation in vitro , 2001, Journal of cellular biochemistry.

[29]  M. E. Adams,et al.  Degeneration and the chemical composition of the human lumbar intervertebral disc , 1987, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[30]  James D. Kang,et al.  New Use of a Three-Dimensional Pellet Culture System for Human Intervertebral Disc Cells: Initial Characterization and Potential Use for Tissue Engineering , 2001, Spine.

[31]  S. Koh,et al.  Histological changes in aging lumbar intervertebral discs. Their role in protrusions and prolapses. , 1990, The Journal of bone and joint surgery. American volume.

[32]  Hideki Yoshikawa,et al.  Bone Morphogenetic Protein Signals Are Required for Cartilage Formation and Differently Regulate Joint Development During Skeletogenesis , 2002, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[33]  F. Luyten,et al.  Mechanisms of GDF-5 action during skeletal development. , 1999, Development.

[34]  U. Liebert,et al.  Stabilization of transgene expression by incorporation of E3 region genes into an adenoviral factor IX vector and by transient anti-CD4 treatment of the host. , 1996, Gene therapy.

[35]  S. O'Kane,et al.  Transforming growth factor βs and wound healing , 1997 .

[36]  S. Trippel.  Growth factor actions on articular cartilage. , 1995, The Journal of rheumatology. Supplement.

[37]  James D. Kang,et al.  Adenovirus‐Mediated Gene Transfer to Nucleus Pulposus Cells: Implications for the Treatment of Intervertebral Disc Degeneration , 1998, Spine.

[38]  R. Ross,et al.  Effects of growth factors in vivo. I. Cell ingrowth into porous subcutaneous chambers. , 1987, The American journal of pathology.

[39]  Joseph A. Buckwalter,et al.  PROTEOGLYCANS OF HUMAN INFANT INTERVERTEBRAL DISC , 1985 .

[40]  E. Thonar,et al.  Quantification of 35S-labeled proteoglycans complexed to alcian blue by rapid filtration in multiwell plates. , 1994, Analytical biochemistry.

[41]  A. Kudo,et al.  Recombinant human growth/differentiation factor 5 stimulates mesenchyme aggregation and chondrogenesis responsible for the skeletal development of limbs. , 1996, Growth factors.

[42]  J. P. Thompson,et al.  Stimulation of Mature Canine Intervertebral Disc by Growth Factors , 1991, Spine.

[43]  P. Guerne,et al.  Growth factor responsiveness of human articular chondrocytes: Distinct profiles in primary chondrocytes, subcultured chondrocytes, and fibroblasts , 1994, Journal of cellular physiology.

[44]  M. Sporn,et al.  Recombinant latent transforming growth factor beta 1 has a longer plasma half-life in rats than active transforming growth factor beta 1, and a different tissue distribution. , 1990, The Journal of clinical investigation.

[45]  J A Buckwalter,et al.  Proteoglycans of human infant intervertebral disc. Electron microscopic and biochemical studies. , 1985, The Journal of bone and joint surgery. American volume.