• Nancy Ruel, Dessislava Z Markova, Sherrill L Adams, Carla Scanzello, Gabriella Cs-Szabo, David Gerard, Peng Shi, D Greg Anderson, Marc Zack, Howard S An, Di Chen, and Yejia Zhang.
• *Department of Orthopedic Surgery, Rush University Medical Center, Chicago, IL †Department of Orthopedic Surgery, Thomas Jefferson University, Philadelphia, PA ‡Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA §Department of Medicine, Section of Rheumatology, Perelman School of Medicine, and Philadelphia Veterans Affairs Medical Center, University of Pennsylvania, Philadelphia, PA ¶Department of Biochemistry, Rush University Medical Center, Chicago, IL ‖Philadelphia Veterans Affairs Medical Center, and Department of Physical Medicine and Rehabilitation, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA; and **Dow Agrosciences, Indianapolis, IN.
• Spine. 2014 Jul 15; 39 (16): 1274-9.

Study DesignThe presence of fibronectin fragments (FN-fs) and the cleaving enzyme, A disintegrin and metalloproteinase domain-containing protein (ADAM)-8 were examined in human intervertebral disc (IVD) tissue in vitro.ObjectiveTo investigate the presence and pathophysiological concentration of FN-fs and their cleaving enzyme, ADAM-8, in the human IVD tissue.Summary Of Background DataThe 29-kDa FN-f has been shown to result in extracellular matrix loss in rabbit IVDs. However, the concentration of this biologically active fragment in the degenerative human IVD tissue has previously not been determined. Furthermore, it is critical to identify the enzyme(s) responsible for FN cleavage in the IVD.MethodsHuman degenerative IVD tissues were removed during spinal surgery. A normal seeming young adult and an infant human cadaveric sample were obtained as controls. Soluble proteins were extracted, and analyzed by Western blotting using antibodies specific for the human FN neoepitope VRAA²⁷¹. A purified 29-kDa FN-f was used to allow estimation of the concentration of FN-fs in the tissues. ADAM-8, a FN-cleaving enzyme, was analyzed by Western blotting and immunostaining.ResultsAll adult IVD tissues contain many FN-f species, but these species were absent from the infant disc tissue. Moderately degenerative discs contained the highest amount of FN-fs; the concentration was estimated to be in the nanomolar range per gram of tissue. ADAM-8, known to cleave FN resulting in the VRAA²⁷¹ neoepitope, was present in the human disc. ADAM-8 primarily localized in the pericellular matrix of the nucleus pulposus tissue, as determined by immunostaining.ConclusionThis is the first report that N-terminal FN-fs are consistently present in IVD tissues from adult subjects. The pathophysiological concentration of these fragments is estimated to be at nanomolar range per gram of IVD tissue. Furthermore, ADAM-8, known to cleave FN, is present at the pericellular matrix of disc cells.

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