Epigenetic modifications associated with ossification of the thoracic ligamentum flavum: The role of DNA methylation in angiogenesis.

Abstract

Thoracic ossification of the ligamentum flavum may cause severe myelopathy. Vascular angiogenesis in normally avascular ligament tissues is central to its pathogenesis; however, the mechanisms remain unclear. Additionally, although DNA methylation is a well-known epigenetic mechanism, its role in ligament ossification has not been previously defined. We investigated the potential involvement of epigenetics and angiogenic factors in the development of thoracic ossification of the ligamentum flavum. Samples were collected from 35 patients with thoracic ossification of the ligamentum flavum who underwent spinal surgery and 12 patients with lumbar spinal canal stenosis or disc herniation without this condition. Tissue sections were used for histological and immunohistochemical studies, whereas primary ligamentum flavum cells were analyzed for DNA methylation and immunoblotting. Bisulfite sequencing was used to analyze DNA methylation and associated pathways. DNA methylation analysis identified vascular endothelial growth factor, fibroblast growth factor, and epithelial adherens junction signaling pathways as being significantly associated with angiogenesis. Vascular-related probes with high methylation rates included vascular endothelial growth factor, cluster of differentiation 34, secreted frizzled-related protein 1, and SRY-box transcription factor 17. These genes were highly expressed in the affected tissues. Immunohistochemistry revealed the presence of vascular endothelial growth factor and cluster of differentiation 34 in mature and hypertrophic chondrocytes at the ossification front, secreted frizzled-related protein 1 and SRY-box transcription factor 17 in mesenchymal cells surrounding the ossification front, and fibroblasts in degenerated ligament regions. Taken together, these findings suggest that neovascular proliferation occurs as a result of ligament degeneration, and that DNA methylation may influence whether these newly formed vessels acquire ossification-promoting properties. DNA methylation changes may be associated with the expression of factors related to ossification. Pathway analysis identified angiogenesis-related signals as significant, highlighting the importance of neovascularization in the process of ossification.