Facilitation of Hematopoietic Reconstitution Via Inhibition of Bone Marrow Endothelial Cell-Mediated SDF-1 Signaling.
Authors: Doan Phuong L. , Russell J. Lauren , Himburg Heather A. , Meadows Sarah K. , Daher Pamela , Helms Katherine , Quarmyne Mamle , Harris Jeffrey R. , Reya Tannishtha , Chao Nelson J. , Kirsch David G. , Chute John P. 
Published: October 2019

Abstract
Abstract 3859
Hematopoietic stem cell (HSC) regeneration is influenced by specialized bone marrow (BM) microenvironments, but the mechanisms that drive HSC regeneration remain incompletely defined. We have recently reported that deletion of the pro-apoptotic proteins, Bak and Bax, in Tie2+ bone marrow endothelial cells (BM ECs)(Tie2Cre;Bak-/-;BaxFl/- mice) caused a significant protection of the BM HSC pool and the BM sinusoidal vasculature in mice following high dose total body irradiation (TBI). We also confirmed that this protection of the BM HSC pool was caused by protection of BM Tie2+ ECs via generation of chimeric mice (Tie2Cre;Bak-/-;BaxFl/- BM; wild type BM ECs) which contained 4.8-fold less BM long-term repopulating HSCs compared to mice bearing deletion of Bak and Bax in both BM HSCs and BM ECs. In order to determine the mechanism through which Tie2+ BM ECs regulate HSC regeneration, we generated primary BM EC lines from Tie2Cre;Bak-/-;BaxFl/- mice and Tie2Cre;Bak-/-;BaxFl/+ control mice. We then compared the capacity for Bak/Bax -/- BM ECs to support BM HSC regeneration in vitro compared to Bak/Bax +/&minus; BM ECs. BM c-kit+sca-1+lin- (KSL) stem/progenitor cells were irradiated with 300 cGy and then placed in 7 day culture with Bak/Bax -/- BM ECs or Bak/Bax +/&minus; BM ECs. Culture with Bak/Bax -/- BM ECs did not yield a significant increase in total viable cells, but yielded 2000-fold increased number of BM KSL cells (p < 0.05, n=3) compared to cultures with Bak/Bax +/&minus; ECs. This significant expansion of phenotypic BM stem/progenitor cells corresponded to a 4-fold increase in CFU-S12 cells in the Bak/Bax -/- EC cultures vs. Bak/Bax +/&minus; EC cultures (p=0.01, n=5). We subsequently compared the level of expression of several microenvironmental ligands which are putatively involved in regulating hematopoiesis. We found that BM ECs from Tie2Cre;Bak-/-;BaxFl/- mice had 37-fold lower expression of stromal-derived factor-1 (SDF-1, CXCL12) compared to BM ECs from Tie2Cre;Bak-/-;BaxFl/+ mice. Moreover, 7 days after TBI, Tie2Cre;Bak-/-;BaxFl/- mice had a 41-fold increase in total viable BM cell counts and had a persistently lower SDF-1 expression on BM ECs (2.7-fold) compared to Tie2Cre;Bak-/-;BaxFl/+ mice (p=0.003). Therefore, we hypothesized that inhibition of SDF-1 signaling might facilitate hematopoietic regeneration following injury. Interestingly, the addition of a blocking anti-SDF1 antibody to cultures of irradiated BM KSL cells with Bak/Bax -/- ECs caused a 50% increase in total cell recovery (p<0.05), a 2.5 fold increase in BM KSL cell recovery (p<0.05) and a 2.2-fold increase in BM CFC recovery (p<0.05) compared to culture with Bak/Bax -/- ECs alone. However, the addition of anti-SDF1 antibody caused a 3-fold decrease in CFU-S12 recovery compared to Bak/Bax -/- EC cultures without anti- SDF1 antibody (p<0.05). Taken together, these data suggest that inhibition of SDF-1 signaling via BM ECs accelerates BM progenitor cell regeneration following injury but is deleterious to the recovery of the BM HSC pool. Targeted therapies aimed at inhibition of SDF-1 signaling may facilitate short-term hematopoietic reconstitution following injury via modulation of BM vascular niche signaling, but this may be at the expense of the BM HSC pool.

Disclosures:
No relevant conflicts of interest to declare.