Heparanase isoform expression and extracellular matrix remodeling in intervertebral disc degenerative disease

Abstract

Objective: To determine the molecules involved in extracellular matrix remodeling and to identify and quantify heparanase isoforms present in herniated and degenerative discs.

Introduction: Heparanase is an endo-beta-glucuronidase that specifically acts upon the heparan sulfate chains of proteoglycans. However, heparanase expression in degenerative intervertebral discs has not yet been evaluated. Notably, previous studies demonstrated a correlation between changes in the heparan sulfate proteoglycan pattern and the degenerative process associated with intervertebral discs.

Methods: Twenty-nine samples of intervertebral degenerative discs, 23 samples of herniated discs and 12 samples of non-degenerative discs were analyzed. The expression of both heparanase isoforms (heparanase-1 and heparanase-2) was evaluated using immunohistochemistry and real-time RT-PCR analysis.

Results: Heparanase-1 and heparanase-2 expression levels were significantly higher in the herniated and degenerative discs in comparison to the control tissues, suggesting a possible role of these proteins in the intervertebral degenerative process.

Conclusion: The overexpression of heparanase isoforms in the degenerative intervertebral discs and the herniated discs suggests a potential role of both proteins in the mediation of inflammatory processes and in extracellular matrix remodeling. The heparanase-2 isoform may be involved in normal metabolic processes, as evidenced by its higher expression in the control intervertebral discs relative to the expression of heparanase-1.

Figure 1

Fibrous annulus and pulposus nucleus HPA1 and HPA2 immunohostochemistry. A, Fibrous annulus control disc HPA1 C20 Santa Cruz primary antibody; B, Fibrous annulus control disc HPA2 C17 primary antibody; C, Fibrous annulus degenerative disc HPA1 C20 Santa Cruz primary antibody; D, Fibrous annulus degenerative disc HPA2 C17 Santa Cruz primary antibody; E, Pulposus nucleus control disc HPA1 C20 Santa Cruz primary antibody; F, Pulposus nucleus control disc HPA2 C17 Santa Cruz primary antibody; G, Pulposus nucleus degenerative disc HPA1 C20 Santa Cruz primary antibody; H, Pulposus nucleus degenerative disc HPA2 C17 Santa Cruz primary antibody. Magnification: 400X.

Figure 2

Analysis of HPA1 and HPA2 immunohistochemical results. The values indicate the expression index (IE) obtained from digital quantification after immunohistochemical reactions using specific HPA1 C20 and HPA2 C17 antibodies (Santa Cruz, Biotechnology, CA, USA) as described in the Methods. ^**P<0.001, Mann-Whitney Test, in comparison with the control IE values.

Figure 3

Quantitative HPA1 and HPA2 expression. Statistical analyses were performed using the Student t-test. *P<0.05, comparison of the control group to the herniated and degenerative disc groups; **P<0.001, statistically significant difference between HPA2 expression in the herniated and degenerative groups.