Angiogenesis in the Outer Membrane of Chronic Subdural Hematomas through Thrombin-Cleaved Osteopontin and the Integrin alpha 9 and Integrin beta 1 Signaling Pathways

Background: A chronic subdural hematoma (CSDH) is considered to be an inflammatory and angiogenic disease. The CSDH outer membrane, which contains inflammatory cells, plays an important role in CSDH development. Osteopontin (OPN) is an extracellular matrix protein that is cleaved by thrombin, generating the N-terminal half of OPN, which is prominently involved in integrin signal transduction. We explored the expression of the N-terminal half of OPN in CSDH fluid and the expression of integrins alpha 9 and beta 1 and the downstream components of the angiogenic signaling pathways in the outer membrane of CSDHs. Methods: Twenty samples of CSDH fluid and eight samples of CSDH outer membrane were collected from patients suffering from CSDHs. The concentrations of the N-terminal half of OPN in CSDH fluid samples were measured using ELISA kits. The expression levels of integrins alpha 9 and beta 1, vinculin, talin-1, focal adhesion kinase (FAK), paxillin, alpha-actin, Src and beta-actin were examined byWestern blot analysis. The expression levels of integrins alpha 9 and beta 1, FAK and paxillin were also examined by immunohistochemistry. We investigated whether CSDH fluid could activate FAK in cultured endothelial cells in vitro. Results: The concentration of the N-terminal half of OPN in CSDH fluid was significantly higher than that in the serum. Western blot analysis confirmed the presence of these molecules. In addition, integrins alpha 9 and beta 1, FAK and paxillin were localized in the endothelial cells of vessels within the CSDH outer membrane. FAK was significantly phosphorylated immediately after treatment with CSDH fluid. Conclusion: Our data suggest that the N-terminal half of OPN in CSDH fluid promotes neovascularization in endothelial cells through integrins alpha 9 and beta 1. The N-terminal half of OPN, which is part of the extracellular matrix, plays a critical role in the promotion of CSDHs.