Abstract
Hematopoietic stem cell (HSC) trafficking is regulated by a number of complex mechanisms. Among them are the transmembrane protein Robo4 and the vascular cell adhesion molecule, VCAM1. Endothelial VCAM1 is a well-known regulator of hematopoietic cell trafficking, and our previous studies revealed that germline deletion of Robo4 led to impaired HSC trafficking, with an increase in vascular endothelial cell (VEC) numbers and downregulation of VCAM1 protein on sinusoidal VECs. Here, we utilized two Robo4 conditional deletion models in parallel with Robo4 germline knockout mice (R4KO) to evaluate the effects of acute and endothelial cell-specific Robo4 deletion on HSC trafficking. Strikingly similar to the R4KO, the acute deletion of Robo4 resulted in altered HSC distribution between the bone marrow and blood compartments, despite normal numbers of VECs and wild-type levels of VCAM1 cell surface protein on sinusoidal VECs. Additionally, consistent with the R4KO mice, acute loss of Robo4 in the host perturbed long-term engraftment of donor wild-type HSCs and improved HSC mobilization to the peripheral blood. These data demonstrate the significant role that endothelial Robo4 plays in directional HSC trafficking, independent of alterations in VEC numbers and VCAM1 expression.
Highlights
The bone marrow (BM) vascular endothelium is known both as a physical barrier to Hematopoietic stem cell (HSC) trafficking and as a scaffolding for HSC residence
We revealed that germline deletion of Robo4 (R4KO) resulted in altered HSC location, increased HSC mobilization efficiency in response to the CXCR4 inhibitor AMD3100, increased vascular permeability, decreased numbers of vascular endothelial cell (VEC) in the BM, and reduced BM sinusoidal VCAM1 expression [16,17]
We hypothesized that increased radiation preconditioning would circumvent the requirement for endothelial Robo4 in promoting HSC engraftment
Summary
The bone marrow (BM) vascular endothelium is known both as a physical barrier to HSC trafficking and as a scaffolding for HSC residence. In contrast to this observation and to our hypothesis that mice lacking Robo4 would exhibit less damage to BM tissue, the radiation experiments demonstrated that R4KO animals had significantly greater loss of total BM cells, non-hematopoietic stromal cells, and VECs compared to the WT cohort (Fig 2B and 2D).
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