Changes in matrix metabolism during intervertebral disc degeneration

Abstract

Gabriella Cs-Szabo, PhD, Deborah Ragasa-San Juan, MS, Vani Turumella, MS, Koichi Masuda, MD, Eugene J. Thonar, PhD, Howard S. An, MD, Chicago, ILIntroduction: Intervertebral disc (IVD) degeneration is associated with loss of matrix molecules and, consequently, a change in the biomechanical properties of the spine. Effective treatment or prevention of degenerative disc disease is not easily achieved because the molecular events that drive these processes are poorly understood. The goal of this study was to follow molecular changes in the annulus fibrosus (AF) and nucleus pulposus (NP) during disc degeneration to identify key players that could be targeted in future intervention. We focused on the following matrix components: (i) aggrecan and versican (large aggregating proteoglycans) along with their binding partner, link protein; (ii) collagens type I and II; and (iii) small proteoglycans (biglycan, decorin and fibromodulin) which can play major roles in matrix repair through their interactions with growth factors, fibronectin and fibrillar collagens.Methods: Four normal human IVD samples from the lumbar spine and 20 degenerated (surgical samples from patients who underwent anterior lumbar discectomy and fusion) IVD samples were used for this study. These IVDs were graded using the MRI based Thompson grading system. AF and NP were separated, and the intermediate tissue from between AF and NP was discarded. Samples were processed for RNA isolation and protein extraction. Semi-quantitative reverse transcription-polymerase chain reaction was used to quantify message levels of matrix components and was normalized to the signal for GAPDH. In addition, the proteins coded by these mRNAs were semi-quantified by comparative Western blotting by measuring the densities of the immunostained bands on the transfer membranes, using an internal control. The message and protein levels of aggrecan, versican, collagens type 1 and 2, biglycan, decorin, fibromodulin and link protein were analyzed as a function of the grade of tissue degeneration.Results: At early stages of degeneration (grades 2-3), the contents of matrix components were significantly elevated in AF (e.g. aggrecan, versican and fibromodulin increased 1.5-fold each, p<0.05). In more severely degenerated tissues (grade 4), levels of large aggregating proteoglycans decreased in AF (aggrecan and versican decreased to 1/3 of grade 2 level, p<0.01), but levels of small proteoglycans (biglycan, decorin and fibromodulin) increased further by 1.5-1.8-fold. Expression of the respective mRNAs showed the same trend. However, in seriously degenerated samples (grade 5), all message and protein levels decreased (eg, collagen type II decreased to of the normal level, p<0.05). In contrast, matrix components showed a continuous decrease by increasing grade of degeneration in NP (eg, in grade 5 tissue decorin content was one tenth, p<.03, and aggrecan one third, p<.05, of the normal). The decrease in content of matrix constituents, in part, was the result of decrease in mRNA expression.Discussion: These results led us to postulate that cells in the AF respond to early degeneration by upregulating biosynthetic processes, thus switching on the repair mechanisms. Decline in the synthesis of aggrecan and collagen type 2 in grade 4 and 5 tissues, however, may lead to complete degeneration of the AF. We further suggest that the consequent increase in the concentrations of small proteoglycans, capable of binding growth factors and influencing collagen fibrillogenesis, might be responsible for the eventual failure of the repair processes in heavily degenerated tissues. Furthermore, the results also suggest that cells in NP are less effective than cells in the AF in repairing damage of the matrix in which those cells reside.