Inhibition of Prostaglandin E2 (PGE2) Signaling by Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) or EP4 Receptor Antagonism Expands Hematopoietic Stem and Progenitor Cells (HSPC) and Enhances Their Mobilization to Peripheral Blood in Mice and Baboons.

Abstract

Abstract 84PGE2 is a physiological regulator of HSPC and exhibits both stimulatory and inhibitory activities. We recently reported that PGE2 increases CXCR4 expression and homing of HSPC to bone marrow (Hoggatt et al, Blood 2009). Since PGE2 is produced within the marrow microenvironment, we hypothesized that blocking PGE2 synthesis in vivo via inhibition of cyclooxygenases (COX) by NSAIDs would facilitate HSPC mobilization and serve as an adjunct to current mobilization regimens. Treatment of mice with the NSAID indomethacin b.i.d. along with G-CSF for 4 days resulted in an increase in peripheral blood (PB) CFU-GM over G-CSF alone (1.96±0.19 fold, p<0.001). Indomethacin treatment for 4 days without G-CSF resulted in marginal, but significant, HSPC mobilization. In addition, NSAID significantly increased mobilization of Sca-1+, c-kit+, Lineageneg (SKL) cells (G-CSF:1565±25 vs G-CSF + NSAID:6447±1324) and the primitive stem cell population of CD150+CD48− SKL cells (G-CSF:57±1 vs G-CSF + NSAID:217±53) per ml blood. Indomethacin synergistically enhanced mobilization by a single administration of AMD3100 alone or in combination with GROβ, indicating G-CSF independent mechanisms. To confirm enhanced mobilization in a non-human primate model, 4 baboons were mobilized with G-CSF + NSAID, or G-CSF alone in a crossover protocol. NSAID co-treatment increased CFU-GM (2845 vs 5975, p=0.03) and CD34+ cells (1061 vs 2201, p=0.03) per ml blood compared to G-CSF alone. Total CFC analysis indicated that NSAID enhanced mobilization of CFU-GM, BFU-E and CFU-GEMM. To determine if long-term repopulating cells were mobilized, lethally irradiated mice were transplanted with 2×106 murine PB mononuclear cells mobilized by G-CSF or G-CSF + NSAID. In mice receiving the G-CSF + NSAID mobilized graft, faster recovery of both neutrophils and platelets was observed (Days to 50%/100% recovery; Neutrophils: G-CSF 19/26 vs G-CSF + NSAID 14/22; Platelets: G-CSF 20/28 vs G-CSF + NSAID 16/24). In murine competitive repopulation assays, increased competitive repopulating units were quantitated in G-CSF + NSAID mobilized grafts compared to G-CSF. Since PGE2 can be produced by COX 1 and COX 2 enzymes, mice were treated with COX 1 specific (SC-560, Valeryl Salicylate), COX 2 specific (Valdecoxib, NS-398) or dual specific (Aspirin, Ibuprofen) NSAIDs with G-CSF. COX 1 specific NSAIDs had no effect on HSPC mobilization, while COX 2 specific and dual inhibitors significantly enhanced mobilization over G-CSF alone. The lipoxygenase (LOX) inhibitor baicalein had no effect on HSPC mobilization and the COX/LOX inhibitor Licofelone was no more effective than NSAIDs, indicating that the mobilization enhancing effect is COX specific and not due to general eicosanoid inhibition. Intriguingly, the dual COX inhibitors produced the greatest activity and a dose-dependent response (max response: 3.92±0.15 fold over G-CSF), indicating inhibition of both COX 1 and COX 2 is required for optimal enhancement. Since PGE2 signals through 4 G-protein coupled receptors (EP1-4), mice were administered EP specific antagonists + G-CSF, or EP specific agonists + NSAID + G-CSF. EP4 antagonists significantly enhanced mobilization over G-CSF alone (2.02±0.21 fold, p<0.005), while an EP1-3 antagonist had no effect. Similarly, a selective EP4 agonist (L-902,688) abrogated NSAID enhancement, while Butaprost (EP2) or Sulprostone (EP1/3) had no effect, indicating that a lack of EP4 receptor signaling is responsible for enhanced HSPC mobilization. Treatment of bone marrow cells with EP specific agonists/antagonists in vitro demonstrates inhibition of colonies by EP4 agonists and a block of inhibition by EP4 antagonists, suggesting a model in which a lack of EP4 signaling drives HSPC expansion. In vivo administration of NSAIDs for 4 days resulted in expansion of CFU-GM and SKL cells per femur, indicating that the mechanism of enhanced mobilization may be in part due to expansion of marrow HSPC. Our results define a novel role for NSAIDs and suggest that the addition of NSAIDs, particularly dual COX inhibitors (i.e. Aspirin or Ibuprofen), to current mobilization regimens may increase HSPC yield. In addition, we have identified novel mechanisms mediated by EP receptors and show that HSPC expansion and mobilization can be enhanced in vivo through antagonism of the EP4 receptor, defining a new pharmaceutical target for hematopoietic mobilization and transplantation.Disclosures: No relevant conflicts of interest to declare.