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 naïve media, ECs exposed to AFC conditioned media expressed higher mRNA and protein levels of key biomarkers of invasive EC phenotype, MMP-2 (2×), MMP-13 (4×), and PDGF-B (1.5-2×), and NGF (24.9±15.2pg/mL vs 0 in naïve media). Treatment of AF cells with EC culture conditioned media decreased collagen type II expression two fold. Considerable quantities of pro-angiogenic factors IL-8 (396.7±302.0pg/mL) and VEGF (756.2±375.9pg/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.
Bibliographical noteFunding Information:
HMEC-1 was kindly provided by Professor Daniel C Devor (Department of Cell Biology and Physiology, University of Pittsburgh). We thank the Dr Rocky Tuan and his laboratory members for providing technical assistance and support for the MMP assays and EC culture work. We gratefully acknowledge Kevin Ngo and Qing Dong for their technical assistance in PCR and IHC measurements. This work was supported in part by NIH grant AG033046 (Nam Vo), NASS Research Traveling Fellowship Award (Hong Joo Moon), and the Albert B. Ferguson, Jr. M.D. Orthopaedic Fund of the Pittsburgh Foundation .
- Annulus fibrosus
- Endothelial cells
- Intervertebral disc degeneration
- Tip cells
ASJC Scopus subject areas
- Biomedical Engineering
- Orthopedics and Sports Medicine