Poster Presentation 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

Introduction

Matrix metalloproteinases (MMPs) are family of zinc-dependent enzymes capable of degrading various components of ECM in diverse homeostatic and pathological processes. The activity of MMPs is regulated by interaction with endogenous inhibitors, tissue inhibitor of metalloproteinases (TIMPs). An altered ratio of MMPs/TIMPs plays an important role in tissue ECM remodeling. The aim of this study was to investigate MMP-2 expression in a rat ligamentum flavum (LF) hypertrophy model and the impact of elastin-derived peptides (EDPs) on TIMPs expression in rat LF cells in vitro.

Methods

To establish the rat LF hypertrophy model, increased stress was induced by surgical destabilization at L3/4 level of the spine. At 6 and 12 weeks after surgical destabilization, rats were sacrificed for histological study with hematoxylin and eosin and elastin staining. Gene expression of MMP-2, MMP-9, TIMP-1 to TIMP-4 were analysis using RT-qPCR. Protein expression of elastin and MMP-2 in rat LF were validated using western blot. To examine the impact of EDPs on MMP-2 and TIMPs expression in rat LF cells, isolated normal rat LF cells were treated with 100µg/ml EDPs, with or without pre-treatment of inhibitors – elastin receptor complex (ERC), chondroitin sulfate A (CSA) and 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA). Expression of MMP-2, TIMP-1 and TIMP-2 were analyzed using RT-qPCR and western blot. Cell culture medium was collected and assayed for MMP-2 using rat MMP-2 ELISA kit.

Results

Spinal destabilization induced LF hypertrophy at 6 and 12 weeks, as evidenced by increased LF thickness and area. Histologically, the hypertrophied LF exhibited significant elastic fibre degradation. In the LF of the destabilized group, mRNA and protein levels of the MMP-2 expression increased. In addition, TIMP-2 and TIMP-3 were upregulated in the destabilized group compared to the sham group. From the in vitro study, EDPs suppressed MMP-2 expression and secretion in rat LF cells, and also reduced the mRNA and protein expression of TIMP-1 and TIMP-2. Interestingly, after EDPs exposure, the calculated ratio of MMP-2/TIMP-1 and MMP-2/TIMP-2 significantly increased and the regulation of MMP-2/TIMPs was in elastin receptor complex (ERC)-dependent manner.

Discussion

Increased activity of MMP-2 had been implicated in the elastic fibre degradation of hypertrophied LF, generating EDPs, which further increased the ratio of MMP-2/TIMPs in LF cells an ERC-dependent manner. However, further studies underpinning the EDPs mechanism of action in the process of LF hypertrophy are needed.