Endothelial Blood-Bone Marrow-Barrier Dynamically Regulates Balanced Stem and Progenitor Cell Trafficking and Maintenance
Authors: Itkin Tomer , Gur-Cohen Shiri , Ludin Aya , Kalinkovich Alexander , Golan Karin , Caglio Giulia , Ornitz David M. , Adams Ralf H. , Kollet Orit , Lapidot Tsvee 
Published: October 2019

Abstract
Abstract 507
Bone marrow (BM) endothelial cells (BMECs) serve as ‘niche’ cells for both hematopoietic and mesenchymal stem and progenitor cells (HSPC/MSPC). Yet BMECs control of HSPC bi-directional trafficking between the BM and peripheral blood (PB) through the blood-bone marrow-barrier (BBMB) is poorly understood.
In vivo treatment with the pro-angiogenic cytokine FGF-2 reduced BM CXCL12 levels, and functionally upregulated CXCR4 expression on primitive Lineage−/Sca-1+/c-Kit+ (LSK) HSPCs accompanied by their increased in vitro CXCL12-induced migration. However, instead of expected HSPC mobilization effect, FGF-2 treatment resulted in reduced numbers of HSPCs in the PB while increasing BM HSPC levels. Based on this, we hypothesized that FGF-2 modifies the BM vascular endothelium, which may be responsible for the observed increase in HSPC retention. Accordingly, we examined BBMB permeability by injecting fluorescently labeled Low Density Lipoprotein (LDL). FGF-2 treatment resulted in reduced BM penetration and incorporation of LDL. Moreover, BM homing of HSPCs was significantly reduced in FGF-2 treated recipients. To further examine endothelial involvement, applying the murine CRE-Lox system for conditional gene knock-out, we used VE-Cadherin-CREERT2 transgenic mice crossed with FGFR1flox/flox/FGFR2flox/flox mice to generate endothelial specific tamoxifen-inducible knock-out of the two endothelial predominantly expressed FGF receptors (eFGFR1/2 KO). FGF-2 treatment of eFGFR1/2 KO mice did not exhibit increased BM retention and reduced homing capabilities of HSPCs as in WT treated mice, suggesting that endothelial specific activation of FGF signaling regulates the BBMB functional control of HSPC bi-directional trafficking. Importantly, eFGFR1/2 KO mice exhibited reduced levels of CD45−/CD11b−/Ter-119−/Sca-1low/-/PDGFβR+ MSPCs together with reduced levels of CD34−LSK and SLAM HSPCs. FGF-2 treatment increased both HSPC and MSPC levels in WT, but not in eFGFR1/2 KO mice. These results imply that hampering endothelial FGF signaling interferes with HSPC/MSPC maintenance, while increasing BBMB permeability. Mechanistically, FGF-2 treatment resulted in decreased MMP-9 protease activity levels in BM supernatants combined with upregulated Timp-1 (an endogenous MMP-9 inhibitor) mRNA levels in total BM cells. Furthermore, FGF-2 treatment reduced eNOS phosphorylation and NO content in total BM cells and in BMECs. As both NO and MMP-9 can promote VE-Cadherin shedding, and subsequently increase endothelial barrier permeability, we observed increased VE-Cadherin expression levels on BMECs following FGF-2 treatment. Supporting this notion, in vivo administration of neutralizing VE-Cadherin antibodies efficiently increased HSPC egress by increasing BBMB permeability. Additionally, VE-Cadherin neutralization increased HSPC BM homing and LDL incorporation. These results reveal that interfering with endothelial adhesion interactions increases HSPC egress and homing. Examination of eNOS KO, Timp-1 KO and WT mice revealed that during steady state, mature WBC counts in the PB were similar. However, PB HSPC numbers were decreased in eNOS KO mice and increased in Timp-1 KO mice as measured by CFU-C and LSK. Similarly, HSPC BM homing capacity and LDL incorporation were decreased in eNOS KO mice and increased in Timp-1 KO mice. These results suggest that NO and MMP-9 mediated shedding of VE-Cadherin promotes BBMB permeability, which regulates egress and homing of immature HSPCs.
In conclusion, our findings reveal that FGF-2-induced expansion of both HSPCs and MSPCs involves BMEC activation. Yet, FGF-stimulated HSPCs fail to egress into the PB and are retained in the BM due to decreased endothelial BBMB permeability. Thus, FGF signaling in BMECs may serve as a “gate-keeper” for HSPC trafficking synchronized with HSPC/MSPC maintenance. We suggest that BMECs are dynamically balanced between their dual role as a ‘niche’, regulating HSPC and MSPC maintenance and as a selective, anatomical barrier regulating HSPC bi-directional trafficking. When the BBMB restricts HSPC trafficking via reduced permeability, it provides better HSPC/MSPC support and maintenance. On the other hand, upon functioning as a trafficking site with increased BBMB permeability, BMEC-provided support and maintenance is reduced.

Disclosures:
No relevant conflicts of interest to declare.