MMP-2 mediates local degradation and remodeling of collagen by annulus fibrosus cells of the intervertebral disc

IntroductionDegeneration of the intervertebral disc (IVD) is characterized by marked degradation and re-structuring of the annulus fibrosus (AF). While several matrix metalloproteinases (MMPs) have been found to be more prevalent in degenerate discs, their coordination and function within the context of the disease process are still not well understood. In this study, we sought to determine whether MMP-2 is associated with degenerative changes in the AF, and to identify the manner by which AF cells utilize MMP-2. Methods: Two established animal models of disc degeneration, static compression and trans-annular needle puncture of rodent caudal discs, were examined for MMP-2 immunopositivity. Using lentiviral transduction of an shRNA expression cassette we screened and identified an effective shRNA sequence for generating stable RNA interference to silence MMP-2 expression in primary rat AF cells. Gelatin films were used to compare gelatinase activity and spatial patterns of degradation between transduced cells, and both non-infected and nonsense shRNA controls. The functional significance of MMP-2 was determined by assessing the ability for cells to remodel collagen gels. Results: Both static compression and 18g annular puncture of rodent caudal discs stimulated an increase in MMP-2 activity with concurrent lamellar disorganization in the AF, while 22g and 26g needle injuries did not. To investigate the functional role of MMP-2, we established lentivirus-mediated RNAi to induce stable knock down of transcript levels by as much as 88%, and protein levels by as much as 95% over 10 days. Culturing transduced cells on gelatin films confirmed that MMP-2 is the primary functional gelatinase in AF cells, and that MMP-2 is utilized locally in regions immediately around AF cells. In collagen gels, transduced cells demonstrated an inability to remodel collagen matrices. Conclusions: Our study indicates that increases in MMP-2 observed in human degenerate discs are mirrored in experimentally-induced degenerative changes in rodent animal models. AF cells appear to utilize MMP-2 in a very directed fashion for local matrix degradation and collagen remodeling. This suggests that MMP-2 may have a functionally significant role in the etiology of degenerative disc disease, and could be a potential therapeutic target.



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