Angiopoietin-2 Promotes Mechanical Stress-induced Extracellular Matrix Degradation in Annulus Fibrosus Via the HIF-1α/NF-κB Signaling Pathway

Abstract

Objective: Mechanical stress is an important risk factor for intervertebral disc degeneration (IVDD). Angiopoietin-2 (ANG-2) is regulated by mechanical stress and is widely involved in the regulation of extracellular matrix metabolism. In addition, the signaling cascade between HIF-1α and NF-κB is critical in matrix degradation. This study aims to investigate the role and molecular mechanism of ANG-2 in regulating the degeneration of annulus fibrosus (AF) through the HIF-1α/NF-κB signaling pathway.

Methods: The bipedal standing mice IVDD model was constructed, and histological experiments were used to evaluate the degree of IVDD and the expression of ANG-2 in the AF. Mouse primary AF cells were extracted in vitro and subjected to mechanical stretching experiments. Western blot assay was used to detect the effect of mechanical stress on ANG-2, and the role of the ANG-2-mediated HIF-1α/NF-κB pathway in matrix degradation. In addition, the effect of inhibiting ANG-2 expression by siRNA or monoclonal antibody on delaying IVDD was investigated at in vitro and in vivo levels. One-way ANOVA with the least significant difference method was used for pairwise comparison of the groups with homogeneous variance, and Dunnett's method was used to compare the groups with heterogeneous variance.

Results: In IVDD, the expressions of catabolic biomarkers (mmp-13, ADAMTS-4) and ANG-2 were significantly increased in AF. In addition, p65 expression was increased while HIF-1α expression was significantly decreased. The results of western blot assay showed mechanical stress significantly up-regulated the expression of ANG-2 in AF cells, and promoted matrix degradation by regulating the activity of HIF-1α/NF-κB pathway. Exogenous addition of Bay117082 and CoCl₂ inhibited matrix degradation caused by mechanical stress. Moreover, injection of neutralizing antibody or treatment with siRNA to inhibit the expression of ANG-2 improved the matrix metabolism of AF and inhibited IVDD progression by regulating the HIF-1α/NF-κB signaling pathway.

Conclusion: In IVDD, mechanical stress could regulate the HIF-1α/NF-κB signaling pathway and matrix degradation by mediating ANG-2 expression in AF degeneration.

Keywords: Angiopoietin-2; Annulus fibrosus; HIF-1α/NF-κB signaling pathway; Intervertebral disc degeneration; Mechanical stress.