499 SUSTAINED RELEASE OF SFLT01 IS NECESSARY AND FEASIBLE FOR PROLONGED INTRAARTICULAR DELIVERY TO THE KNEE TO NEUTRALIZE VEGF LOCALLY

498 – Figure 1. NOx (LEFT) and PGE2 (RIGHT) production by AF cells from 4 degenerative human tissue samples after treatment with control media or media with IL-17, IFNγ, or both IL-17 and IFNγ. The values for the media control only were indicated as dotted horizontal lines. (control), TNFα (25 ng/mL), IL17 (10 ng/mL), IFNγ (200 U/mL) or both IL17 and IFNγ. After 72 hours, the supernatant was evaluated for release of nitric oxide (NOx, Griess reaction) and prostaglandin E2 (PGE2, ELISA). One-way ANOVA and post-hoc Dunn’s analysis evaluated differences amongst treatment groups for NOx and PGE2 release (p=0.05). Results: Stimulation of AF cells with IL17 or IFNγ alone did change NOx levels from control values; however, a significant variability in NOx responses was observed across human subjects (Figure 1 left). In the presence of IFNγ costimulant, IL17 induced average 2-fold increase in NOx production above that of IL17 alone (IL17: average NOx = 22 μM, IL17 + IFNγ: NOx = 45 μM, Figure 1 left). In contrast, stimulation with IL17 alone led to a significant elevation in PGE2 levels in all 4 samples (average 14fold increase, Figure 1 right). As for the NOx result, co-stimulation with IL17 and IFNγ led to a large increase in PGE2 release, more than 24-fold over control values (Figure 1 right, IL17 + IFNγ PGE2 release = 1.36 ng/ml; Control PGE2 release = 0.06 ng/ml). In general, the induction of NOx and PGE2 release by IL17 and IFNγ co-stimulation was significantly larger than that due to TNFα stimulation, for which few changes in NOx and a 10-fold increase in PGE2 was observed. Conclusions: The results of this study demonstrate that degenerative human IVD cells may respond to IL17 stimulation with increased production of the inflammatory mediators, PGE2 and NOx, with a more robust effect observed in the presence of IFNγ. Degenerative and herniated IVD explants are known to contain IFNγ, and IFNγ is known to act synergistically with IL17 to promote release of inflammatory mediators in other cell types. This work demonstrates that the responsiveness of IVD cells to IL17 and IFNγ is consistent with other cell types, and suggests a potential role for these cytokines in contributing to IVD pathology.