title: 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.
abstract: 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 ± 15.2 pg/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 ± 302.0 pg/mL) and VEGF (756.2 ± 375.9 pg/mL) were also detected in the conditioned media of untreated AF cell culture.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Ferguson Laboratory for Orthopaedic Research, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA; Department of Neurosurgery, College of Medicine, Korea University, Seoul, Republic of Korea.
authors.forename: H J
authors.initials: HJ
authors.lastname: Moon
authors.affiliation: Ferguson Laboratory for Orthopaedic Research, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA; Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Yurube
authors.affiliation: Department of Orthopaedic Surgery, Center for Cellular and Molecular Engineering, University of Pittsburgh School of Medicine, 450 Technology Drive, Pittsburgh, PA 15219, USA.
authors.forename: T P
authors.initials: TP
authors.lastname: Lozito
authors.affiliation: Ferguson Laboratory for Orthopaedic Research, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
authors.forename: P
authors.initials: P
authors.lastname: Pohl
authors.affiliation: Ferguson Laboratory for Orthopaedic Research, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
authors.forename: R A
authors.initials: RA
authors.lastname: Hartman
authors.affiliation: Ferguson Laboratory for Orthopaedic Research, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA; Department of Physical Medicine and Rehabilitation, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors.forename: G A
authors.initials: GA
authors.lastname: Sowa
authors.affiliation: Ferguson Laboratory for Orthopaedic Research, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA. Electronic address: kangjd@upmc.edu.
authors.forename: J D
authors.initials: JD
authors.lastname: Kang
authors.affiliation: Ferguson Laboratory for Orthopaedic Research, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA. Electronic address: von@upmc.edu.
authors.forename: N V
authors.initials: NV
authors.lastname: Vo
mesh_terms: D000328:Adult; D002196:Capillaries / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D024043:Collagen Type II / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000494:pharmacology*; D042783:Endothelial Cells / Q000187:drug effects; D004730:Endothelium, Vascular / Q000378:metabolism*; D005109:Extracellular Matrix / Q000473:pathology*; D005260:Female; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis; D007403:Intervertebral Disc / Q000098:blood supply / Q000294:innervation*; D055959:Intervertebral Disc Degeneration / Q000378:metabolism* / Q000473:pathology; D008297:Male; D045726:Metalloproteases / Q000378:metabolism; D008875:Middle Aged; D009389:Neovascularization, Pathologic / Q000378:metabolism; D020932:Nerve Growth Factor / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis
chemical_list: D024043:Collagen Type II; D017077:Culture Media, Conditioned; D016209:Interleukin-8; D010982:Platelet-Derived Growth Factor; D042461:Vascular Endothelial Growth Factor A; D020932:Nerve Growth Factor; D045726:Metalloproteases
keywords: Angiogenesis; Annulus fibrosus; Endothelial cells; Intervertebral disc degeneration; Metalloproteinases; Tip cells