Ageing affects chondroitin sulfates and their synthetic enzymes in the intervertebral disc

Abstract

The depletion of chondroitin sulfates (CSs) within the intervertebral disc (IVD) during degenerative disc disease (DDD) results in a decrease in tissue hydration, a loss of fluid movement, cell apoptosis, a loss of nerve growth inhibition and ultimately, the loss of disc function. To date, little is known with regards to the structure and content of chondroitin sulfates (CSs) during IVD ageing. The behavior of glycosaminoglycans (GAGs), specifically CSs, as well as xylosyltransferase I (XT-I) and glucuronyltransferase I (GT-I), two key enzymes involved in CS synthesis as a primer of glycosaminoglycan (GAG) chain elongation and GAG synthesis in the nucleus pulposus (NP), respectively, were evaluated in a bovine ageing IVD model. Here, we showed significant changes in the composition of GAGs during the disc ageing process (6-month-old, 2-year-old and 8-year-old IVDs representing the immature to mature skeleton). The CS quantity and composition of annulus fibrosus (AF) and NP were determined. The expression of both XT-I and GT-I was detected using immunohistochemistry. A significant decrease in GAGs was observed during the ageing process. CSs are affected at both the structural and quantitative levels with important changes in sulfation observed upon maturity, which correlated with a decrease in the expression of both XT-I and GT-I. A progressive switch of the sulfation profile was noted in both NP and AF tissues from 6 months to 8 years. These changes give an appreciation of the potential impact of CSs on the disc biology and the development of therapeutic approaches for disc regeneration and repair.

Figure 1

Schematic of CS synthesis [adapted from Laboratories K. KEGG Pathway Maps: Glycosaminoglycans Synthesis, Japan, 2013]. The CS results from the polymerization of a succession of glycans on the serine residue of the Asn-X-Ser/Thr peptidic motif via different enzymatic reactions. The enzymes are represented in green, while the protein core is represented in orange.

Figure 2

Gross morphologies of 6-month-old (1), 2-year-old (2), and 8-year-old (3) bovine caudal intervertebral discs. A decrease of the NP/AF ratio is observed upon ageing.

Figure 3

(1) Representative images of nucleus pulposus tissue stained with (a–c) Safranin O/ fast green (FCF), (d–f) for XT-I, and (g–i) for GT-I in 6-month-old (a, d and g), 2-year-old (b, e and h) and 8-year-old (c, f and i) bovine IVD tissues (n=5; scale bar=100 μm). (2) Quantification of the XT-I and GT-I staining intensity in NP tissue. Data were normalized to the surface area and are represented as the mean±s.e.m. (n=5). * denotes significant differences between the different groups at P<0.05.

Figure 4

(1) Representative images of AF tissue stained with (a–c) Safranin O/FCF; (d–f) for XT-I and (g–i) for GT-I in 6-month-old (a, d and g), two year-old (b, e and h), and 8-year-old (c, f and i) bovine IVD tissues (n=5; scale bar=100 μm). (2) Quantification of XT-I and GT-I staining intensities in AF tissue. Data were normalized to the surface area and are represented as the mean±standard error of the mean (n=5). * denotes significant differences between the different groups at P<0.05.

Figure 5

Age and tissue-related changes of (a) total sGAG content of bovine IVD quantified by the DMMB assay and (b) sulfated CS content quantified by HPLC. Data were normalized to the DNA content and are represented as the mean±s.e.m. (n=5). * represents significant differences at P<0.05.

Figure 6

Quantities of C0S, C4S and C6S disaccharides (μg/μg of DNA) in 6-month, 2-year and 8-year-old bovine IVD tissues. Data were normalized to the DNA content and are represented as the mean±s.e.m. (n=5); * represents significant differences at P<0.05 for C0S, C4S, C6S and the total CSs.

Figure 7

Percentages of disaccharides C0S, C4S and C6S in 6-month-old, 2-year-old and 8-year-old bovine (a) NP and (b) AF tissues. Data were normalized to the DNA content and are represented as the mean±s.e.m. (n=5); * represents significant differences at P<0.05.