The effects of human intervertebral disc aggrecan on neuronal and endothelial cell growth

Abstract

Introduction The development of ‘discogenic’ low back pain has been associated with increased nerve growth into degenerated intervertebral discs, particularly in neovascularized areas. A marked loss of proteoglycans, particularly from the disc's inner regions, is also a marked feature of the degenerative process. As proteoglycans from other tissues can have inhibitory effects on nerve growth, we have hypothesized that the alterations in proteoglycan content seen in disc degeneration may affect the growth of nerves and blood vessels into the disc. To test our hypothesis, we have established cell culture assays to determine the effects of human intervertebral disc aggrecan, which forms the major proteoglyan found in the disc, on neuronal and endothelial cell growth.Materials and methods Aggrecan (A1D1 preparations) isolated from the outer (anulus fibrosus, AF) and inner (nucleus pulposus, NP) regions of human lumbar intervertebral discs was incorporated into culture substrata, using methods previously described (Snow et al. 1990). Chick dorsal root ganglia (DRG) and the cell line SH‐SY5Y were used as models of nerve growth. HMEC‐1 and Eahy‐926 cell lines were used as models of endothelial cell growth.Results Human disc aggrecan inhibited SHSY‐5Y cell attachment, SHSY5Y neurite outgrowth and induced sensory DRG neurite growth cone turning in a concentration‐dependent manner. Sensory neurites emanating from DRG across permissive substrates (collagen/laminin) were repelled by high (1 mg/ml), but not low (0.01 mg/ml) concentrations of disc aggrecan and became aligned to the aggrecan border. Disc aggrecan similarly inhibited endothelial cell adhesion, cell spreading and migration. HMEC‐1 and Eahy926 cells migrated over collagen substrates (type I) until they encountered disc aggrecan, where they either stopped migrating or, more commonly, changed their direction of movement and aligned to the aggrecan border. In general, aggrecan isolated from the AF was more inhibitory than that isolated from the NP. The inhibitory effects were partially abrogated following enzymic deglycosylation of the aggrecan, or if aggrecan was added in solution (i.e. was nonsubstrate bound).Conclusions This study provides evidence that disc aggrecan inhibits neuronal and endothelial growth and migration and therefore supports a hypothesis that a loss of aggrecan from degenerated discs predispose the tissue to vascular and neuronal invasion.