Vav1 Regulates Perivascular Homing, Bone Marrow Retention and Engraftment of Hematopoietic Stem Cells Via SDF1a Signaling

Abstract 400

Engraftment and maintenance of hematopoietic stem and progenitor cells (HSPC) depends on extracellular signals present in specialized microenvironments in the bone marrow (BM). Osteoblasts, endothelium, and nestin-positive mesenchymal cells (N+MC), potentially contribute to the hematopoietic microenvironment (HM) and HSPC “niche” through membrane and secreted molecules such as stem cell factor, CXCL12/SDF1α, and integrin ligands. The crucial intracellular pathways that coordinate these signals, and thus regulate HSPC localization and interaction with components of their niche, remain poorly understood. We have implicated Rho GTPases Rac1 and Rac2 in the regulation of HSPC trafficking and engraftment (Gu et al. Science, 2003; Cancelas et al. Nat Med, 2005). Rho GTPases are activated by c-kit, CXCR4 and β1 integrin membrane receptors via largely undefined guanine nucleotide exchange factors (GEF). We hypothesized that the hematopoietic-specific GEF Vav1 regulates HSPC engraftment and retention by controlling Rho GTPase activation in response to signals from the HM and studied the hematopoietic phenotype and function of Vav1-/- HSPC mice. Analysis of the frequency of circulating progenitors in Vav1^-/- mice revealed unexpected alterations in HSPC trafficking, including significantly decreased numbers of circulating CFC, absence of circadian fluctuation, and impaired progenitor mobilization induced by AMD3100 (AMD3100 mobilized 925+/−200 vs. 420+/−300 CFC/ml in WT vs Vav1-/-, P<0.01). Total leukocyte counts were similar in both genotypes after AMD3100 treatment, showing that this change was specific for primitive cells. In addition, GCSF-induced mobilization was also defective in Vav1-/- mice (640+/−40 CFC/ml vs 105+/−40 CFC/ml in WT vs Vav1-/-; P<0.01). These processes depend on SDF1α signalling, suggesting that in the absence of Vav1, HSPC were insensitive to induced changes in SDF1α levels or activity in the BM. Confirming that Vav1 was specifically required for SDF1α-mediated chemotactic responses, Vav1^-/- HSPC demonstrated defective linear migration in a SDF1α gradient in vitro (net path length 53+/−10μm vs 27+/−4μm in WT vs Vav1-/- cells, P<0.01); however, Vav1 was not required for baseline or SDF1α -induced β1 integrin-mediated adhesion. These changes were associated with a lack ofagonist-dependent increase in Rac/Cdc42 activation after treatment with SDF-1α and an unexpectedly high baseline Rac/Cdc42 activity level. The downstream effector PAK-1 showed the same abnormal activation profile. To investigate the role of this pathway in in vivo engraftment and retention in the HM, we performed repopulation assays and determined the localization of transplanted Vav1^-/- HSPC in the BM by intravital microscopy. Deletion of Vav1 was associated with nearly complete loss of HSPC engraftment in short-term (50% donor WT chimerism vs 0% Vav1-/- at 1 month) and long-term (56% vs 0%, WT vs Vav1-/- at 4 months) assays. Microscopically, Vav1^-/- HSPC localized normally with respect to osteoblasts and the endosteal matrix.

(Col2.3-GFP transgenic recipients), but showed significantly impaired localization to the proximity of SDF1α-secreting, N+MC (nestin-GFP+ recipients), 1 hr after transplant. At this early timepoint, only 6.25% of Vav1^-/- HSPC, compared with 45.8% of WT HSPC, were located at less than 30 μm from a nestin⁺ cell. Considering only HSPC with nestin⁺ cells in their proximity, the mean distance of HSPC to the nearest nestin⁺ cell was significantly (p=0.005) shorter for WT vs Vav1^-/- HSPC. This early abnormal localization was associated with significantly decreased retention of Vav1-/- HSPC (44-60% relative to WT) at 48 hours post-transplant in the BM cavities of both nestin-GFP and Col2.3-GFP recipient mice; this alteration in numbers of HSPC was not related to alterations in HSPC proliferative status or survival. In addition to previously characterized roles in immune responses, Vav1 functions as a critical molecule in HSPC, where it is specifically required for Rac/Cdc42 activation, SDF1α-dependent perivascular post-transplant homing, BM retention and engraftment. Our data define a pathway differentially regulating HSPC positioning with respect to N+MC, and support the concept that localization in the proximity of this perivascular niche component is important in the initial stages of post-transplant HSPC homing and is required for their early retention.