Institutional members access full text with Ovid®

Fibronectin Fragments and the Cleaving Enzyme ADAM-8 in the Degenerative Human Intervertebral Disc

Ruel, Nancy PhD*; Markova, Dessislava Z. PhD; Adams, Sherrill L. PhD; Scanzello, Carla MD, PhD§; Cs-Szabo, Gabriella PhD; Gerard, David MS; Shi, Peng DDS, PhD*; Anderson, D. Greg MD; Zack, Marc MS**; An, Howard S. MD*; Chen, Di MD, PhD; Zhang, Yejia MD, PhD*,‖

doi: 10.1097/BRS.0000000000000397
Basic Science

Study Design. The 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.

Objective. To investigate the presence and pathophysiological concentration of FN-fs and their cleaving enzyme, ADAM-8, in the human IVD tissue.

Summary of Background Data. The 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.

Methods. Human 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.

Results. All 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.

Conclusion. This 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.

Level of Evidence: N/A

We have shown that fibronectin fragments (FN-fs) with molecular weights of about 29 kDa are present in degenerative intervertebral discs (IVDs), at an estimated concentration in the nanomolar range per gram of tissue. Furthermore, a FN-cleaving enzyme, A disintegrin and metalloproteinase domain-8, is also present in the degenerative human IVD.

*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.

Address correspondence and reprint requests to Yejia Zhang, MD, PhD, Philadelphia Veterans Affairs Medical Center, and Department of Physical Medicine and Rehabilitation, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA; E-mail: yejia.zhang@uphs.upenn.edu

Acknowledgment date: January 6, 2014. Revision date: March 13, 2014. Acceptance date: April 4, 2014.

The manuscript submitted does not contain information about medical device(s)/drug(s).

National Institute of Child Health and Human Development (NICHD, 1K08 HD049598-01) funds were received to support this work.

Relevant financial activities outside the submitted work: consultancy, grants/grants pending, royalties, and Medical Advisory Board.

Nancy Ruel and Dessislava Z. Markova contributed equally to the work.

© 2014 by Lippincott Williams & Wilkins