Engraftment and Reconstitution of Hematopoiesis Is Dependent on Endothelial Cell Expressed Prostaglandin EP4 Receptor Signaling Mediated Regeneration of Vascular Niche

Defining the cellular and molecular components of the bone marrow (BM) niche that regulate the homeostasis and regeneration of hematopoietic stem and progenitor cells (HSPCs) can facilitate targeted interventions to improve hematopoietic recovery after injury and enhance stem cell engraftment after transplantation. We have recently shown that BM niche prostaglandin E2 (PGE2) signaling via the EP4 receptor is crucial for hematopoietic reconstitution, as transplantation of wild-type donor cells into EP4 receptor deficient recipient mice shows reduced stem cell engraftment. However, the specific contribution of EP4 signaling on different niche constituents in hematopoietic regeneration is unknown. To investigate which niche constituent/stromal population EP4 signaling functionally contributes to hematopoietic regeneration, we utilized genetic mouse models, in which EP4 could be deleted in specific stromal cell types.

We conditionally deleted EP4 expression in nestin expressing mesenchymal stem cell (MSC), leptin receptor expressing mesenchymal stromal cell or tie 2 expressing endothelial cell (EC) invivo using a cre-recombinase approach. At steady-state, selective deletion of EP4 from nestin⁺ MSC (nestin-cre EP4^flox/flox mice), leptin receptor⁺stromal cell (leptin R-cre EP4^flox/flox mice) or tie 2⁺EC (tie 2-cre EP4^flox/flox mice) showed no change in BM MSC (CD45^-Ter119^-CD31^-CD51⁺PDGFR⁺), leptin receptor⁺stromal cells (CD45^-Ter119^-leptin R⁺) and EC (CD45^-ter119^-CD31⁺VE-cadherin⁺) number and survival. We next determined whether loss of EP4 expression/signaling in stromal cell specific genetic knockout mouse models affects BM niche constituents after exposure to ionization radiation. Interestingly, loss of EC but not nestin/leptin EP4 expression severely disrupted the BM vasculature and reduced EC and MSC survival at 24 hours post total body lethal (TBI) irradiation, compared to wild-type irradiated mice, and was associated with reduced expression of Survivin and increased expression of Bax, anti-apoptotic and pro-apoptotic factors respectively. In addition, niche EC and MSC regeneration at 2-month post irradiation was substantially reduced in tie 2-cre EP4^flox/flox mice compared to wild-type mice. Nestin-cre EP4^flox/flox mice and leptin R-cre EP4^flox/flox niche restoration was equivalent to wild-type mice. To determine whether loss of EP4 receptor in different niche cells differentially regulates hematopoietic regeneration, we transplanted wild-type donor cells into nestin-cre EP4^flox/flox or tie 2-cre EP4^flox/flox recipient mice. Chimeric mice made with EC-specific EP4 deficient stroma showed reduced HSPC engraftment compared to chimeric mice made with wild-type stroma. In contrast, stem cell engraftment in chimeric mice made with MSC-specific EP4 deficient stroma was not reduced. In addition, in vivo administration of PGE2 into wild-type recipient mice at six-hour post irradiation enhanced donor cell engraftment. However, PGE2 treatment failed to enhance donor cell engraftment into tie 2-cre EP4^flox/flox recipient mice. These data suggest that EC EP4 expression is important for niche restoration and hematopoietic regeneration after transplantation. To identify the mechanism involved in EP4 signaling mediated niche restoration, we measured angiocrine factors in the BM niche of lethally irradiated wild-type and tie 2-cre EP4^flox/flox mice. Interestingly, we found that vascular endothelial growth factor (VEGF) expression was substantially reduced in the BM of tie 2-cre EP4^flox/flox mice compared to wild-type mice. To investigate whether EP4 signaling regulates niche restoration by stimulating VEGF expression/production, we treated lethally irradiated tie 2-cre EP4^flox/flox mice with recombinant VEGF (20 ng/mouse/day) for 3 days. In vivo administration of recombinant VEGF substantially enhanced niche restoration in tie 2-cre EP4^flox/floxmice compared to control tie 2-cre EP4^flox/flox. In conclusion, our study suggests that EC specific EP4 receptor signaling-mediated increased expression of VEGF promotes vascular niche restoration after myelosuppression and supports hematopoietic regeneration.