Effects of secreted factors in culture medium of annulus fibrosus cells on microvascular endothelial cells: elucidating the possible pathomechanisms of matrix degradation and nerve in-growth in disc degeneration

Objective: To test whether the interaction between annulus fibrosus cells (AFCs) and endothelial cells (ECs) disrupts matrix homeostasis and stimulates production of innervation mediators. Methods: Human microvascular ECs were cultured in the conditioned media of AF cell culture derived from degenerated human surgical specimen. Matrix-metalloproteinases (MMPs) and platelet-derived growth factor (PDGF) of ECs of this culture were analyzed- by qRT-PCR, Western, and immunofluorescence. Vascular endothelial growth factor (VEGF), Interleukin-8 (IL-8), and nerve growth factor (NGF) in the media of this cell culture were assayed by ELISA. To determine the effects of ECs on AFCs, qRT-PCR was performed to determine mRNA levels of collagen I, II and aggrecan in AFCs cultured in EC conditioned media. Results: Compared to ECs cultured in na ve media, ECs exposed to AFC conditioned media expressed higher mRNA and protein levels of key biomarkers of invasive EC phenotype, MMP-2 (2x), MMP-13 (4x), and PDGF-B (1.5-2 x), and NGF (24.9 15.2 pg/mL vs 0 in na ve media). Treatment of AF cells with EC culture conditioned media decreased collagen type II expression two fold. Considerable quantities of proangiogenic factors IL-8 (396.7 302.0 pg/mL) and VEGF (756.2 375.9 pg/mL) were also detected in the conditioned media of untreated AF cell culture. Discussion: AFCs from degenerated discs secreted factors which stimulated EC production of factors known to induce matrix degradation, angiogenesis, and innervation. IL-8 and VEGF maybe the secreted factors from AFCs which mediate a pro-angiogenic stimulus often implicated in the development of disc degeneration. Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International.