HSPC Mobilization Research Articles

Endothelial Blood-Bone Marrow-Barrier Dynamically Regulates Balanced Stem and Progenitor Cell Trafficking and Maintenance

AbstractAbstract 507Bone 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.

Coagulation Factor Thrombin Regulates Hematopoietic Stem and Progenitor Cell Egress and Mobilization Via PAR-1 & CXCR4 Upregulation, SDF-1 Secretion and EPCR Shedding

Abstract 2341Hematopoietic stem and progenitor cell (HSPC) egress from the bone marrow (BM) to the circulation is tightly regulated and is accelerated during stress conditions. The G-protein-coupled receptor protease-activated receptor-1 (PAR-1) and its activator thrombin play an important role in coagulation following injury and bleeding. We report that a single injection of thrombin induced rapid HSPC mobilization within one hour, increasing circulating leukocytes, predominantly CFU-C and primitive Lin−/Sca-1+/c-Kit+ (SKL) progenitor cells. This rapid mobilization was preceded by a dramatic decrease of SDF-1 (CXCL12) in BM stromal cells, including rare Nestin+ mesenchymal stem cells (MSC) which functionally express PAR-1 and release SDF-1. Thrombin injection also increased expression of PAR-1 and CXCR4 by BM HSPC. These results suggest involvement of the coagulation cascade of thrombin & PAR-1 in rapid SDF-1 secretion from niche supporting BM stromal cells as part of host defense and repair mechanisms. Administration of a PAR-1 specific antagonist (SCH79797) upregulated BM SDF-1 levels and significantly reduced the amounts of circulating CFU-C and primitive SKL progenitor cells. In vitro stimulation of BM mononuclear cells with thrombin for 1 hour led to increased CXCR4 expression by Lin−/c-Kit+ progenitors, accompanied by enhanced spontaneous and SDF-1 induced migration. Of note, specific PAR-1 inhibition in vitro significantly reduced SDF-1-directed migration of Lin-/c-Kit+ progenitors. Mechanistically, we found that thrombin - activated PAR-1 induced the downstream p38 MAPK and eNOS (nitric oxide synthase) signaling pathways. Long term repopulating hematopoietic stem cells (HSC) in murine BM highly express endothelial protein C receptor (EPCRhigh) (Balazs & Mulligan et al Blood 2006; Kent & Eaves et al Blood 2009). EPCR is expressed primarily on endothelial cells (EC) and has anti coagulation and anti inflammatory roles. Surface EPCR expression on EC is downregulated by many factors, including PAR-1 activation by thrombin, a process which is termed shedding and is not fully understood. Importantly, we found that over 90% of BM CD45+/EPCRhigh long-term HSC express PAR-1 and that circulating primitive HSPC in the blood and spleen lack EPCRhigh expression. In addition, in-vivo thrombin administration downregulated EPCR from BM HSC via eNOS signaling, thus allowing the release of stem cells from their BM microenvironment anchorage to the circulation. Correspondingly, in eNOS deficient mice, thrombin failed to induce PAR-1 upregulation, EPCR shedding, and HSPC mobilization. Recently, we reported that the antioxidant NAC inhibits G-CSF induced mobilization (Tesio & Lapidot et al Blood 2011). Co-administration of G-CSF with NAC prevented PAR-1 upregulation, concomitantly with reduced HSPC mobilization and increased levels of EPCRhigh HSC in the BM. Treatment of PAR-1 antagonist with G-CSF inhibited PAR-1 and CXCR4 upregulation on BM leukocytes and immature Lin−/c-Kit+ cells accompanied by increased levels of BM EPCRhigh HSC and reduced HSPC mobilization. Tissue factor (TF) is the main initiator of the coagulation system via the formation of an enzymatic “prothrombinase complex” that converts prothrombin to active thrombin. Unexpectedly, we found a unique structure of cell clusters expressing TF, located preferentially in the trabecular-rich area of the femoral metaphysis in murine bone tips, a region highly exposed to osteoclast/osteoblast bone remodeling. In vitro, immature osteoclasts exhibited increased TF expression in cell fusion areas, suggesting that in vivo osteoclast maturation activates the coagulation thrombin/PAR-1 axis of HSPC migration to the circulation. Finally, mimicking bacterial infection a single injection of Lipopolysaccharide (LPS), rapidly and systemically upregulated TF in the murine BM. LPS treatment prompted an increase in thrombin generation and subsequently HSPC mobilization, which was blocked by the PAR-1 antagonist. In conclusion, our study reveals a new role for the coagulation signaling axis, which acts on both hematopoietic and stromal BM cells to regulate steady state HSPC egress and enhanced mobilization from the BM. This thrombin/PAR-1 signaling cascade involves SDF-1/CXCR4 interactions, immature osteoclast TF activity, Nestin+/PAR-1+ MSC secretion of SDF-1 and EPCR shedding from hematopoietic stem cells. Disclosures:No relevant conflicts of interest to declare.

Role for vitamin D receptor in the neuronal control of the hematopoietic stem cell niche

AbstractHematopoietic stem/progenitor cells (HSPCs) are released from the bone marrow to the circulation by the cytokine, granulocyte colony-stimulating factor, via sympathetic nervous system (SNS)–mediated osteoblast suppression. Because the orientation of HSPCs in their osteoblastic niche is reported to be guided by [Ca2+], we speculated on a cooperation between the calcium-regulating hormones and SNS in the regulation of HSPC trafficking. Here, we present the severe impairment of granulocyte colony-stimulating factor–induced osteoblast suppression and subsequent HSPC mobilization in vitamin D receptor (VDR)–deficient mice. In osteoblasts, functional VDR possessing, at least in part, a transcriptional activity, was specifically induced by β2-adrenergic receptor (AR) agonists. While β2-AR agonists transiently increased mRNA expression of Vdr and its downstream gene, Rankl, 1α,25-dihydroxyvitamin-D3 sustained the β2-AR–induced Rankl expression at high level by stabilizing VDR protein. These data suggest that VDR is essential for durable β2-AR signaling in the stem cell niche. Our study demonstrates not only a novel function of VDR as a critical modulator of HSPC trafficking, but also the presence of a SNS-mediated, bone-remodeling mechanism through VDR. VDR contributes to brain-bone-blood integration in an unanticipated way distinct from other classical calcium-regulating hormones.

MT1-MMP Associates with Membrane Lipid Rafts and Is Required for G-CSF-Induced Hematopoietic Stem/Progenitor Cell Mobilization.

Abstract 3536Poster Board III-473Hematopoietic stem/progenitor cells (HSPC) that have been mobilized from bone marrow (BM) to peripheral blood (PB) by granulocyte-colony stimulating factor (G-CSF) are being used for autologous and allogeneic transplantation. However, the molecular mechanisms of HSPC mobilization are not completely understood. The key molecules and interactions that regulate HSPC mobilization include various adhesion molecules, chemokine stromal cell-derived factor (SDF)-1 and its receptor CXCR4, and proteases including the soluble matrix metalloproteinase (MMP)-9. Membrane type (MT)-1 MMP, which is localized on the leading edge of migrating cells, has strong pericellular proteolytic activity, activates the latent MMPs especially proMMP-2, and has been implicated in mediating migration of tumor cells, monocytes, endothelial as well as CD34+ HSPC. MT1-MMP not only degrades several extracellular matrix molecules in the pericellular space, but also cleaves cell surface molecules such as CXCR4 and CD44, cytokines, and chemokines including SDF-1. In this study we focused on characterizing the role of MT1-MMP during G-CSF-induced migration, its regulation and subcellular localization in HSPC and mature cells. We found that MT1-MMP mRNA and protein expression (as determined by RT-PCR and flow cytometry) in G-CSF-mobilized mature hematopoietic cells (monocytes and neutrophils) as well as immature CD34+ cells was significantly higher than in their steady-state BM counterparts. Moreover, G-CSF stimulation (i) upregulated MT1-MMP transcription (RT-PCR) and protein synthesis (flow cytometry, Western blot, and confocal microscopy) in BM MNC and CD34+ cells but not in BM stromal cells; and (ii) increased their trans-Matrigel chemoinvasion towards an SDF-1 gradient which was inhibited by the MT1-MMP inhibitor epigallocatechin 3-gallate, by anti-MT1-MMP mAb, and by siRNA silencing of MT1-MMP. To determine the effect of high MT1-MMP expression in hematopoietic cells on the BM microenvironment we co-cultured steady-state BM CD34+ cells with BM fibroblasts. Zymographic analysis of the cell-conditioned media revealed that activation of proMMP-2 occurs only when the co-cultures were stimulated with G-CSF indicating that upregulation of MT1-MMP in CD34+ cells is necessary for proMMP-2 activation as media conditioned by CD34+ cells (silenced with MT1-MMP siRNA) co-cultured with stromal cells did not show proMMP-2 activation. We next focused on determining the signaling pathways that regulate MT1-MMP expression and localization in hematopoietic cells including HSPC during G-CSF-induced migration. We found that although G-CSF activated both phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways (Western blot), upregulation of MT1-MMP by G-CSF, and proMMP-2 activation were PI3K-dependent. Moreover, we demonstrated for the first time that G-CSF incorporated MT1-MMP to membrane lipid rafts of hematopoietic cells in a PI3K-dependent manner since inhibition of this axis by PI3K inhibitor LY290042 reduced MT1-MMP incorporation, an effect not observed with the MAPK inhibitor PD98059. We further demonstrated that by disrupting raft formation using the cholesterol sequestering agent methyl-beta-cyclodextrin, PI3K phosphorylation was inhibited. Subsequently MT1-MMP incorporation into lipid rafts was abrogated resulting in reduced both proMMP-2 activation and HSPC trans-Matrigel migration. We conclude that G-CSF-induced upregulation of MT1-MMP and its incorporation into membrane lipid rafts of hematopoietic cells contributes to the activation of proMMP-2 and to the generation of a highly proteolytic microenvironment in BM, which facilitates egress of HSPC into circulation. Our results suggest that manipulating MT1-MMP expression could become a new strategy to enhance mobilization of HSPC and improve the outcome of transplantation. Disclosures:No relevant conflicts of interest to declare.

BIO5192, a small molecule inhibitor of VLA-4, mobilizes hematopoietic stem and progenitor cells

AbstractHere we show that interruption of the VCAM-1/VLA-4 axis with a small molecule inhibitor of VLA-4, BIO5192, results in a 30-fold increase in mobilization of murine hematopoietic stem and progenitors (HSPCs) over basal levels. An additive affect on HSPC mobilization (3-fold) was observed when plerixafor (AMD3100), a small molecule inhibitor of the CXCR-4/SDF-1 axis, was combined with BIO5192. Furthermore, the combination of granulocyte colony-stimulating factor (G-CSF), BIO5192, and plerixafor enhanced mobilization by 17-fold compared with G-CSF alone. HSPCs mobilized by BIO5192 or the combination of BIO5192 and plerixafor mobilized long-term repopulating cells, which successfully engraft and expand in a multilineage fashion in secondary transplantation recipients. Splenectomy resulted in a dramatic enhancement of G-CSF–induced mobilization while decreasing both plerixafor- and BIO5192-induced mobilization of HSPCs. These data provide evidence for the utility of small molecule inhibitors of VLA-4 either alone or in combination with G-CSF or AMD3100 for mobilization of hematopoietic stem and progenitor cells.

Circadian Traffic of Hematopoietic Stem Cells Is Orchestrated by the Molecular Clock and Mediated by β3 Adrenergic Signals from the Sympathetic Nervous System.

Under homeostasis, low numbers of haematopoietic stem cells (HSPC) are detectable in the bloodstream of mammals. Here, we show the mechanisms behind the robust circadian oscillations of circulating HSPC in mice. In normal 12h light-12h darkness cycles (LD) there were 2–4-fold fluctuations in Lin-Sca-1+c-kit+ cells and competitive repopulating stem cell units (CRU; 16 weeks) between the peak (5h after the initiation of light) and trough (12h later). Circadian fluctuations of HSPC were entrained by photic cues since the pattern was dramatically altered when mice were subjected to continuous light or a “jet lag” of 12h. Because CXCL12 mediates HSPC migration, we evaluated whether it is subjected to circadian control. Indeed, CXCL12 protein and mRNA levels in the bone marrow (BM) fluctuate under all light conditions in antiphase with circulating HSPC. Since the sympathetic nervous system (SNS) affects G-CSF-induced HSPC mobilization (Cell 2006; 124:407–21), we analyzed its role in circadian HSPC release. SNS disruption by injection of 6-hydroxydopamine significantly hampered HSPC egress from the BM, as revealed by the marked reductions in the number of CRU in the blood obtained from sympathectomized animals compared to control mice. Moreover, unilateral section of the sciatic and femoral nerves revealed that the circadian oscillations of Cxcl12 were severely altered in the denervated tibiae but not in the contralateral sham-operated limbs, suggesting the requirement of adrenergic signals locally delivered in the BM. To dissect further the underlying mechanism, we treated BM stromal cells (MS-5) with adrenergic agonists and antagonists. Norepinephrine and isoproterenol, a non-selective β-adrenergic agonist, reduced CXCL12 production in a dose-dependent manner. Unexpectedly, this effect was mediated by the β3- (Adrb3) but not the β2-adrenergic receptor (Adrb2) since it was induced by a β3-adrenergic agonist and inhibited by a specific β3 antagonist while Adrβ2 engagement or blockade had no effect. Similar results were obtained in primary myeloid BM cultures. Further, isoproterenol treatment significantly reduced Cxcl12 expression in stroma derived from Adrb2−/− but not Adrb3−/− mice. In addition, isoproterenol administration increased circulating HSPC in wild-type but not Adrb3−/− mice in which HSPC homing was blocked by inactivation of endothelial selectins and α4 integrins. Since SNS signals can modulate osteoblast proliferation via peripheral expression of clock genes, we profiled the expression of core clock genes in the BM, and found altered patterns of Clock, Bmal1, Per1, Per2, Cry1 and Rev-erb-α under continuous light or jet lag conditions. However, treatment of stromal cells obtained from the BM of Bmal1−/− or Per1−/−Per2m/mmice with isoproterenol reduced Cxcl12 to the same extent as in wild-type control stroma, suggesting that peripheral clock genes do not directly regulate Cxcl12. The physiological oscillations in circulating HSPC may impact the harvested stem cell yield since more HSPC were obtained at the acrophase in mice injected with G-CSF or AMD-3100. All together, these results demonstrate that the cyclical HSPC release and Cxcl12 BM expression are regulated by the central and not the peripheral molecular clock through signals from the SNS that are transduced locally by the β3-adrenergic receptor in BM stromal cells. Since osteoblasts are reported to express only Adrb2, these results also suggest the contribution of other stromal components in the physiological release of HSPC.

Safety and efficacy of combining ATRA with G-CSF in HSPC mobilization; a pilot study in multiple myeloma and non-Hodgkin's lymphoma patients

Safety and efficacy of combining ATRA with G-CSF in HSPC mobilization; a pilot study in multiple myeloma and non-Hodgkin's lymphoma patients