Sequential dynamics of matrix metalloproteinases, tumor necrosis factor–α, vascular endothelial growth factor and plasmin expressions in the resorption process of herniated disc

Abstract

Purpose of study: Granulation tissues of herniated disc (HD) are composed of marked infiltration macrophages and neovascularization that is not observed in the healthy intervertebral disc. Magnetic resonance imaging (MRI) study has shown that epidurally displaced HD tissues more commonly exhibited a gradual decrease in the size of HD. We previously showed that infiltrating macrophages, neovascularization induced by vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMP)-3 and MMP-7 have been implicated in this resorption process with the study of an in vitro coculture model reproducing the acute HD phase. In this report, we examine the interaction of those molecules by investigating their sequential expressions of mRNA at an early time point in that coculture system with semiquantitative reverse transcriptase polymeric chain reaction (RT-PCR).Methods used: Activated murine macrophages were obtained by intraperitoneal administration of thioglycollate 3% medium. Cells were harvested by peritoneal lavage 4 days after, and coccygeal intervertebral disc tissues were obtained. Whole disc tissues or peritoneal macrophages were incubated alone or cocultured. Every 3 hours after cell culture started, total RNA was extracted, and by RT-PCR we examined the semiquantitative expression of MMP-3, -7, VEGF, TNF-α, plasminogen activator tissue type (t-PA) and urokinase type (u-PA), plasmin and GAPDH. Then we analyzed the change of mRNA expression and the interaction of those molecules.of findings: In both singly and cocultured macrophages, MMP-7 and VEGF were expressed constitutively. In cocultured macrophages, TNF-α was expressed at 3 hours and u-PA was expressed at 6 hours. They were changed to weak expression after 24 hours. Plasmin and MMP-3 were detected constitutively after 12 hours. In disc tissues, MMP-3 was expressed before culture and was strongly expressed in cocultured disc tissues at 3 hours; then both singly and cocultured the expression was detected constitutively. u-PA and plasmin were expressed at 3 hours and changed to weak expression after 24 hours. TNF-α was expressed at 6 hours and changed to weak expression after 18 hours. VEGF was not expressed in singly cultured disc tissues. However, VEGF was expressed at 3 hours after coculture, and after 12 hours the expression was detected constitutively. MMP-7 could not be detected in disc tissues. t-PA did not change expression at each time point in each condition.Relationship between findings and existing knowledge: Activated macrophages are known to generate proinflammatory cytokines, such as TNF-α, which is a potent inducer of various MMPs and also a potential regulator of angiogenesis by inducing of VEGF. VEGF also induces PAs, resulting in the generation of plasmin. In turn, plasmin activates MMPs. The activation of MMPs resulting from VEGF induction may result in macrophage infiltration of HD. These factors therefore affected each other and were upregulated in the mechanism of the resorption process of HD. In our cocultured macrophages, TNF-α was first upregulated and then u-PA was expressed. After that, both plasmin and MMP-3 were upregulated. We can conclude a putative mechanism that after the contact between disc tissues and macrophages, coculture condition initiated TNF-α upregulated expression. TNF-α could accelerate both angiogenesis and matrix degrading.Overall significance of findings: In our coculture system, TNF-α was first upregulated after the beginning of coculture. TNF-α could be the key molecule to accelerate the cascade of angiogenesis and disc matrix degradation.Disclosures: No disclosures.Conflict of interest: No conflicts.