Proteomics analysis of the human intervertebral disc reveals potential fibrotic events in degeneration

Abstract

Intervertebral disc (IVD) degeneration is a world-wide health problem and its aetiology is multifactorial with genetic and environmental factors. The IVD is a highly specialised niche, and contains a nucleus pulposus (NP) rich in proteoglycans (PG) and water, surrounded by a collagenous annulus fibrosis (AF). During IVD degeneration, there is a loss of disc matrix, water, and cells from the NP which results in structural changes to the disc, ultimately altering its function and can lead to low back pain. In this study, we systematically analysed changes in the matrix proteins in relation to aging and IVD degeneration. Ethics was approved by the IRB, and clinical IVD samples were obtained with patient consent. The AF and NP were dissected from degenerated and non-degenerated samples. Proteins were extracted with 4M GuHCl buffer, fractionated using CsCl density gradient ultracentrifugation, and separated into soluble/insoluble fractions. Proteins were trypsin digested, labeled with iTRAQ reagents for quantitative analysis using LCMS/MS, and identified using ProteinPilot 4.0. Seven controls were compared to identify age-related changes. In the AF, collagen type I (soluble fraction) and types II and XI (soluble and insoluble fractions) showed a decreasing trend with age. In the NP, type II collagen had a decreasing trend, but in younger controls, was accompanied with increased type I collagen, suggesting a shift in collagen composition. An increasing trend with age was observed for PGs, including decorin, lumican and PRELP (AF and NP), as well as biglycan and mimecan in NP. Seven degenerated samples were compared to controls. Many identified proteins in the degenerated samples showed similar patterns with the controls, indicating that aging and degeneration may involve similar processes. Younger, degenerated samples had elevated levels of lumican, type II collagen, and particularly type I collagen in the NP (insoluble). Older, degenerated IVD had increased aggrecan, decorin and PRELP in the AF (insoluble fraction). Aging and degeneration shared some similarities, as well as differences in the static protein profile. Reduced collagen during degeneration may correlate to decreased tensile strength and fibrotic appearances of degenerated tissues. The differing proteins may aid in understanding the processes involved in aging and degeneration. Further studying of the dynamics of IVD cells in protein production may be achieved with stable isotope labeling by amino acids in cell culture (SILAC), which would identify actively synthesised proteins and further elucidate the cellular functions in the IVD.