Spatial and Temporal Fluctuations In Marrow SDF-1 Following Radiation Injury Regulate Megakaryocyte-Vascular Niche Interactions and Circulating Platelet Levels

The development of megakaryocytes (MKs) in the bone marrow progresses spatially from the endosteal niche, which promotes MK progenitor proliferation, to the sinusoidal vascular niche, the site of terminal maturation and thrombopoiesis. The chemokine SDF-1 (CXCL12), signaling through receptor CXCR4, is produced by stromal cell populations throughout the marrow and is implicated in the maturational chemotaxis of MKs to the sinusoids. Understanding the regulation of MK localization has significance not only for optimal platelet production and the development of therapies for thrombocytopenia, but also in light of the recently proposed role for MKs in supporting hematopoietic stem cells (Heazlewood et al. 2013). In the injury setting of lethal total body irradiation (TBI), it was observed that radioresistant mature MKs relocate to the endosteal niche (Dominici et al. 2009, Olson et al. 2013). Complicating the study of marrow niches post-TBI is the vascular dilation that accompanies the drastic loss of marrow cells. Having confirmed that MKs relocate to the endosteum in our model of sublethal radiation-induced thrombocytopenia (4Gy TBI), we asked whether this localization is due to changes in the spatial distribution of the vasculature or to altered microenvironmental SDF-1. In agreement with other TBI models, we find a significant elevation in SDF-1 transcript levels in the marrow at days 1-3 following 4Gy TBI. Radioresistant MKs, which do not decrease in number until after 3 days, have significantly increased CXCR4 surface expression, a finding we also observe following SDF-1 stimulation of MKs both in vitro and in vivo. In situ hybridization was used to localize the spatial distribution of SDF-1 RNA in femoral marrow. At 2 days post-4Gy, a significant SDF-1 gradient develops with 30% higher SDF-1 message adjacent to the endosteum than in the central marrow. However, this gradient is dynamically eliminated 24 hours later at 3 days post-TBI. These shifts in SDF-1 expression are accompanied by parallel changes in the spatial distribution of MKs by immunohistochemistry. At 2 days post-TBI, there is over a 40% increase in MK in the endosteal niche. In contrast, MKs in the endosteal niche decrease by more than 15% at 3 days, coincident with a significant increase in the MKs associated with vascular endothelium. Thus, these data suggest that the spatial distribution of MKs is dependent upon the localization of SDF-1 in the rapidly fluctuating post-injury bone marrow. To determine if SDF-1 functionally contributes to MK niche changes, we stabilized endogenously-produced SDF-1 using Diprotin A, an inhibitor of SDF-1-inactivating protease DPP4. In uninjured marrow, Diprotin A treatment causes over a 30% rise in MK association with vasculature and a 20% increase in circulating platelets 24 hours later, with no change in MK number. Elevation of vascular SDF-1 by intravenous (IV) administration yields similar results. These data indicate that an endogenous SDF-1 gradient toward the vasculature contributes to homeostatic megakaryopoiesis and thrombopoiesis. At 2 days post-TBI, when endosteal SDF-1 message is increased, stabilization with Diprotin A results in a 40% decrease in MKs associated with vasculature and a small but significant decrease in platelets 24 hours later. Further supporting a role for altered SDF-1 gradients, elevating vascular levels with IV SDF-1 at 2 days causes the opposite effect of Diprotin A, with more MKs found in the vascular niche and a rise in peripheral platelet count. In contrast, at 3 days post-TBI, stabilization of endogenous SDF-1 with Diprotin A causes a further 25% increase in MKs in the vascular niche and a 10% rise in circulating platelets, consistent with the rapid loss of the endosteal SDF-1 gradient. Taken together, our data demonstrate that changes in microenvironmental SDF-1 regulate the spatial distribution of MKs in the post-TBI bone marrow. Importantly, the observed SDF-1 changes have functional consequences for platelet production, as the movement of MKs toward the endosteum decreases circulating platelets, while MK association with the vasculature increases circulating platelets. This knowledge will ultimately lead to improved therapeutic strategies to enhance platelet output in the setting of thrombocytopenia and highlights the need to carefully optimize the timing of therapeutic interventions. Disclosures: No relevant conflicts of interest to declare.