The role of matrix metalloproteinases and vascular endothelial growth factor in the resorption process of herniated disc

Abstract

Purpose of study: Spontaneous resorption of herniated disc (HD) is frequently detected by magnetic resonance imaging. The tendency of HD to spontaneously resorb is directly proportional to the degree of Gd-DTPA enhancement suggesting a vascularization-mediated process that also correlates with prominent macrophage infiltration. Both infiltrating macrophages and factor VIII–positive cells were abundant in epidurally displaced HD, suggesting neovascularization process in HD resorption. We developed an in vivo coculture system of macrophages and disc tissues, which could reproduce the acute phase of HD. Vascular endothelial growth factor (VEGF) is an endothelial-specific mitogen and plays an essential role in the formation of new blood vessels and vascular tubule formation. Currently, we have used this coculture system to investigate the role of MMPs and VEGF in angiogenesis in herniated disc resorption.Methods used: Murine coccygeal intervertebral discs were isolated from mice. Murine macrophages were obtained after intraperitoneal administration of thioglycollate. The isolated murine discs and macrophages were then either cultured separately or in direct contact as cocultures. The conditioned media from the various cultures and cocultures were subjected to reverse transcriptase polymerase chain reaction (RT-PCR) to reveal the cell source of MMP-3, MMP-7, and VEGF and western blotting analysis for determination of MMPs and VEGF expression. To assess the angiogenetic activity of coculture system, we used angiogenesis assay (TCS Biologicals), which can be used to study new blood formation and vascular tubule formation.of findings: Cocultures of disc tissue and macrophages produced elevated levels of MMP-3, MMP-7 and VEGF as determined by western blotting analysis. However, weak expression of them was observed in the single-culture of macrophage or disc tissue. RT-PCR revealed that the majority of the MMP-7 and VEGF were produced by the macrophages, and MMP-3 was by disc chondrocytes under coculture conditions, but both cell types produced detectable levels of all. CD31 (PECAM-1)–positive endothelial cells and formation of anastomosing tubule network were abundant in media derived from coculture compared with single culture.Relationship between findings and existing knowledge: Our study demonstrated that coculture condition resulted in disc degradation mediated by MMP upregulation. In addition, our results suggest that the interaction of macrophages with disc tissues leads to VEGF induction with a stimulation of new blood formation. VEGF is also known to promote monocyte chemotaxis. Thus, VEGF could be essential both for macrophage infiltration into HD tissues, subsequent angiogenesis and HD resorption.Overall significance of findings: In the resorption process of herniated disc, proteoglycan enzyme MMPs and a potent angiogenetic factor VEGF play a pivotal role in degrading disc matrix. These factors may be possible candidates for clinical use as a chemonucleolysis for patients with herniated disc.Disclosures: No disclosures.Conflict of interest: Hirotaka Haro, grant research support: Ministry of Education, Japan.