Figure 2.

15-epi-LXA₄ up regulates SPM and decreases pro-inflammatory mediators in healthy and diseased tendon stromal cells. Tendon stromal cells were derived from healthy hamstring (H, n = 7 donors) or diseased supraspinatus tendons (TD, n = 6 donors). Cells were incubated with 15-epi-LXA₄ (0.1 or 10 nM) or Vehicle for 24 h at 37 °C then with IL-1β (10ngml^—1) for 24 h. (A,B) LM were identified and quantified using LM profiling (see methods for details). Cumulative levels (A) DHA-derived (RvD, PD, MaR) n-3 DPA-derived (RvD_{n-3 DPA}, PD_{n-3 DPA}, MaR1_{n-3 DPA}), EPA-derived (RvE) and AA-derived (LX) pro-resolving mediator levels. (B) Pro-inflammatory eicosanoids (PG, Tx) in tendon stromal cells from healthy volunteers (HV) and patients with tendinopathy (TD). Statistically significant differences were calculated using pairwise Mann-Whitney U tests. *p < 0.05 vs Vehicle incubations. (C) Incubation of IL-1β stimulated diseased tendon cells in 0.1 or 10 nM 15-epi-LXA₄ induced expression of ALOX15 mRNA relative to vehicle controls. (D) Immunocytochemistry for ALOX15 in IL-1β stimulated diseased tendon stromal cells incubated in 10 nM 15-epi-LXA₄. (E) mRNA expression of PDPN, STAT-1 and IL-6 determined using quantitative RTPCR. Gene expression is normalized to β-actin, bars show median values. (F) ELISA assay of IL-6 protein secretion from IL-1β stimulated diseased tendon cells incubated in the presence and absence of 10 nM 15-epi-LXA₄. Data are shown as means and SEM, n = 4 separate donors. (G) Representative immunofluorescence images showing staining for STAT-1 (green), IL-6 (red), PDPN (green), and nuclei (cyan) in IL-1β stimulated diseased tendon stromal cells incubated in 10 nM 15-epi-LXA₄. All images are representative of n = 3 donors. Scale bar, 20 μm.