ab17_2

Cathepsin G in degenerating and healthy discal tissue

Y.T. Konttinen¹, ², ³, E. Kääpä¹, M. Hukkanen⁴, X.H. Gu⁴, M. Takagi⁵, S. Santavirta⁶, H. Alaranta⁷, T.F. Li¹,⁶, A. Suda⁸

¹Institute of Biomedicine, Department of Anatomy, University of Helsinki, Finland; ²Division of Rheumatic Diseases, 4th Dept. of Medicine, Helsinki University Central Hospital; ³ORTON Research Institute, Invalid Foundation, Helsinki, Finland; ⁴Department of Histochemistry, Royal Postgraduate Medical School, Hammersmith Hospital, London, UK; ⁵Department of Restorative Neuromuscular Surgery and Rehabilitation, Tohoku University, Sendai, Japan; ⁶Department of Orthopaedics and Traumatology, Helsinki University Central Hospital, Helsinki, Finland; ⁷Institute of Rehabilitation for Spinal Injuries, Helsinki, Finland; ⁸Department of Orthopedic Surgery, Yamagata University School of Medicine, Yamaga, Japan.

ABSTRACT
Objectives
To assess the eventual presence, tissue localization, molecular forms, amount and activity of cathepsin G in the annulus fibrosus.

Methods
Normal non-autolytic disc tissue was collected from cadavers within 6 hours after death. Degenerate disc samples were collected from low back pain patients undergoing anterior interbody fusion due to severe, discographically verified and painful disc degeneration, and from the posterior parts of intervertebral discs from 10 patients undergoing microscopic discoidectomy because of intervertebral herniation. Avidin-biotin-peroxidase complex staining of cathepsin G was quantitated by morphometry. Cellular localization was analyzed using double immunofluorescence staining of cathepsin G and CD68, proline 4-hydroxylase or von Willebrand factor. Neutral salt extracts were analyzed by using synthetic cathepsin G substrate in spectrophotometry, dot-immunoblotting and Western blotting.

Results
Histological and morphometric image analysis showed increased cellularity, increased numbers of cathepsin G positive cells and neovascularization in degenerated discs compared to control discs. Neutral salt extract of disc tissue, degenerated or normal, in contrast to control material from synovial capsular tissue, did not contain measurable cathepsin G activity, although immunoreactive enzyme was detected in dot-immunoblotting. Western blotting demonstrated that the discal cathepsin G had an apparent molecular weight of 27 kDa.

Conclusion
Due to its properties and localization in normal and pathologically altered tissue, cathepsin G probably plays both a direct and an indirect role in extracellular matrix degradation in the annulus fibrosus. Extracted cationic cathepsin G was immunoreactive, but was functionally inhibited by serpins or, more likely, by polyanionic proteoglycans and saccharins derived from the connective tissue matrix of the annulus fibrosus.

Key words
Cathepsin G, intervertebral disc, angiogenesis.

Supported by grants from the Yrjö Jahnsson Foundation, the Siviä Kosti Foundation, the Invalid Foundation, CIMO and the Finnish Academy.

Please address correspondence and reprint requests to: Y.T. Konttinen, MD, PhD, Institute of Biomedicine, Department of Anatomy, P.O. Box 9, Siltavuorenpenger 20 A, University of Helsinki, FIN-00014 Helsinki, Finland.