Influence of rabbit notochordal cells on symptomatic intervertebral disc degeneration: anti-angiogenic capacity on human endothelial cell proliferation.

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Influence of rabbit notochordal cells on symptomatic intervertebral disc degeneration: anti-angiogenic capacity on human endothelial cell proliferation under hypoxia W.-K. Kwon, H.J. Moon, T.-H. Kwon, Y.-K. Park, J.H. Kim Osteoarthritis and Cartilage Volume 25, Issue 10, Pages 1738-1746 (October 2017) DOI: 10.1016/j.joca.2017.06.003 Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Schematic drawing of the experimental process. Human NP cells were incubated for 6 days and then separated into cultures under two different oxygen concentrations (3% and 21%). After 72 h, the conditioned medium of NP cells was collected to produce NP cell conditioned medium (A). As the second step, ECs were cultured within NPCM with or without NC-rNC coculture, to evaluate the effect of rNC on ECs (B). Ctrl, control; EC, endothelial cell; Exp, experimental. Osteoarthritis and Cartilage 2017 25, 1738-1746DOI: (10.1016/j.joca.2017.06.003) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Expression of angiogenic activator molecules after culture of ECs with and without NC coculture, under two different oxygen concentrations. The VEGF level in EC cells cultured in NPCM was significantly higher under hypoxia (3%) compared to normoxia. Under hypoxic conditions, the expression level of VEGF was significantly lower when ECs were cocultured with NCs (A). VCAM (B) and IL-8 (C) did not show any significant differences in expression level under either oxygen concentration. The dots represent the mean data of three duplication sets of each independent experiments (n = 8). The long horizontal bar represents the mean value and the short horizontal bars represent 95% confidence intervals. Ctrl, control; EC, endothelial cell; Exp, experimental. Osteoarthritis and Cartilage 2017 25, 1738-1746DOI: (10.1016/j.joca.2017.06.003) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Western blotting results of FAK and F-actin expression after NC coculture under hypoxia. When the ECs were cocultured with NCs under hypoxia, both FAK and F-actin expression were significantly inhibited, compared to the EC alone experimental group. Ctrl, control; EC, endothelial cell; Exp, experimental; kDa, kilodaltons. Osteoarthritis and Cartilage 2017 25, 1738-1746DOI: (10.1016/j.joca.2017.06.003) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Immunofluorescence assay results of PDGF expression after NC coculture under hypoxia. When the ECs were cocultured with NCs under hypoxia, the level of PDGF expression was significantly lower than that in the EC alone experimental group. Ctrl, control; EC, endothelial cell; Exp, experimental. Osteoarthritis and Cartilage 2017 25, 1738-1746DOI: (10.1016/j.joca.2017.06.003) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Wound healing migration assay results. Mean cell counts 12 and 24 h after scratch formation. Representative figure of the wound healing migration assay under hypoxia (A) and normoxia (B). The cell counts at 24 h after scratch formation suggested the inhibition of endothelial migration by NC coculture (C). However, no significant effect was seen under normoxia (D). The dots represent the mean data of three duplication sets of each independent experiments (n = 8). The long horizontal bar represents the mean value and the short horizontal bars represent 95% confidence intervals. Ctrl, control; EC, endothelial cell; Exp, experimental. Osteoarthritis and Cartilage 2017 25, 1738-1746DOI: (10.1016/j.joca.2017.06.003) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Schematic summary of our results indicating the anti-angiogenic effect of NCs on ECs, showing the three steps corresponding to the intervertebral disc endothelial angiogenesis flow chart. Osteoarthritis and Cartilage 2017 25, 1738-1746DOI: (10.1016/j.joca.2017.06.003) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions