Bone Marrow Sinusoidal Integrity Regulates Hematopoietic Stem and Progenitor Cell Trafficking

Bone marrow sinusoidal endothelial cells (ECs) are an integral component of the hematopoietic stromal microenvironment and have been implicated in hematopoietic stem and progenitor cell (HSPC) homeostasis, though their role in regulation of hematopoietic trafficking remains uncertain. In this study, we demonstrate that bone marrow (BM) sinusoidal ECs actively regulate HSPC retention and enforced egress out of BM quite surprisingly by serving as “gatekeepers” of HSPC within the marrow niche, a non-paradigmatic mechanism independent of traditional adhesive/chemotactic mechanisms.

Treatment with G-CSF (100 µg/kg, 4 days) resulted in down-regulated expression of VE-cadherin (50±3.3% reduction) and CD31 (35±2.1%) along the gap junctions connecting ECs (CD45^neg Ter119^- VEGFR3⁺) forming BM sinuosoids, reducing cell-cell contact and allowing for increased HSPC trafficking to the peripheral blood. To validate the functional contribution of ECs in HSPC trafficking, mouse (C166 cells) or human (HUVEC) endothelial monolayers were treated with G-CSF for 24 hours in vitro. Treatment of EC monolayers with G-CSF increased dextran-FITC permeability across EC monolayers and increased migration of HSPCs across endothelium, suggesting that a component of G-CSF mobilization activity is the “opening” of EC gap junctions to allow successful HSPC migration through the EC layer. To confirm this increase in endothelial monolayer permeability in response to G-CSF treatment, we measured EC cell-cell contacts both in vitro and in vivo. Immunofluorescence analysis of HUVECs demonstrated significant VE-cadherin+ cell-cell contact in our cultured HUVEC monolayers with a significant loss of cell-cell contact after G-CSF exposure. Histological examination of femur sections from G-CSF treated mice showed a similar disruption of cell-cell contact of VEGFR⁺and Dil-Ac-LDL sinusoids.

Previous reports have demonstrated that pharmacologic antagonism of the dipeptidyl peptidase CD26, or its gene deletion, results in reduced HSPC egress in response to G-CSF. As CD26 is highly expressed on BM ECs, and expression dynamically changes in response to G-CSF treatment, we hypothesized that sinusoidal ECs expressing CD26 may regulate sinusoidal integrity and HSPC egress. Blockade of CD26 activity, or gene deletion of CD26 in ECs, completely reversed the G-CSF-mediated reduction in EC cell-cell junctions accompanied by reduced trans-endothelial migration of HSPC. Given that CD26 is an enzyme that exerts physiologic effects by N-terminal cleavage of effector proteins, we performed a systematic protein sequence search for proteins containing putative CD26 recognition sites. Intriguingly, the neurotransmitter neuropeptide Y (NPY) can be cleaved by CD26, and the full length and cleaved versions have differing functions on NPY receptors expressed on EC. While HSPC mobilization in response to G-CSF was reduced in the absence of CD26 activity, mobilization was restored by treating mice with the cleaved NPY (NPY_3-36) and was inhibited by antagonizing NPY2 and Y5 receptors on ECs, suggesting that reduced mobilization in CD26 knockout mice is caused by a lack of cleaved NPY, preventing an opening of EC cell-cell junctions and allowing for HSPC egress.

If ECs are acting as active “gatekeepers” of HSPC within the marrow space, regulated by CD26 and NPY, we hypothesized HSPC egress in response to agents other than G-CSF would also depend on EC cell-cell junction opening. Interestingly, LPS administration, a mimic of bacterial infection, and AMD3100, a CXCR4 antagonist, also reduced VE-cadherin and CD31 expression along BM sinusoidal EC junctions coincident with enhanced HSPC mobilization. Our results identify an unappreciated active role for bone marrow niche ECs in maintaining HSPC within the marrow space, and describe a heretofore unknown CD26-mediated EC-neuropeptide Y axis regulating optimal HSPC trafficking. These results further refine our knowledge of hematopoietic trafficking mechanisms and identify potential new pharmaceutic targets to regulate trafficking in homeostatic and stress conditions.