Bone Marrow Sinusoidal Endothelial Cells Research Articles

Silencing endomucin in bone marrow sinusoids improves hematopoietic stem and progenitor cell homing during transplantation.

Efficient homing of infused hematopoietic stem and progenitor cells (HSPCs) into the bone marrow (BM) is the prerequisite for successful hematopoietic stem cell transplantation. However, only a small part of infused HSPCs find their way to the BM niche. A better understanding of the mechanisms that facilitate HSPC homing will help to develop strategies to improve the initial HSPC engraftment and subsequent hematopoietic regeneration. Here, we show that irradiation upregulates the endomucin expression of endothelial cells in vivo and in vitro. Furthermore, depletion of endomucin in irradiated endothelial cells with short interfering RNA (siRNA) increases the HSPC-endothelial cell adhesion in vitro. To abrogate the endomucin of BM sinusoidal endothelial cells (BM-SECs) in vivo, we develop a siRNA-loaded bovine serum albumin nanoparticle for targeted delivery. Nanoparticle-mediated siRNA delivery successfully silences endomucin expression in BM-SECs and improves HSPC homing during transplantation. These results reveal that endomucin plays a critical role in HSPC homing during transplantation and that gene-based manipulation of BM-SEC endomucin in vivo can be exploited to improve the efficacy of HSPC transplantation.

Bone Marrow Sinusoidal Endothelial Cells Are a Site of Fgf23 Upregulation in Murine ß-Thalassemia

Fibroblast growth factor-23 (FGF23) is a bone-derived hormonal regulator of mineral metabolism with emerging functions in the bone marrow. FGF23 has been reported to negatively regulate erythropoiesis [PMID:24509850] and promote hematopoietic progenitor mobilization from the bone marrow [PMID:33512467]. Elevated FGF23 levels in the circulation have been described in iron-deficient subjects and also in subjects with the iron-loading anemia β-thalassemia. Recently, we reported bone marrow sinusoidal endothelial cells (BM-SEC) as a novel site of Fgf23 upregulation in mice with chronic iron-deficiency anemia and acute, phlebotomy-induced anemia [PMID:37417950]. We and others have detected Fgf23 mRNA elevation in total bone marrow of Hbb th3/+ mice, a model of non-transfusion dependent β-thalassemia that exhibits systemic iron loading. In the present study, we sought to clarify sites of Fgf23 promoter activity in Hbb th3/+ bone marrow using a reporter allele in which the enhanced green fluorescent protein (eGFP) coding sequence is knocked into the endogenous Fgf23 locus ( Fgf23 eGFP). We generated Hbb +/+ and Hbb th3/+ female littermates that carried either one ( Fgf23 +/eGFP) or zero ( Fgf23 +/+) copies of the Fgf23 reporter allele, which were raised on a standard rodent diet (200 mg/kg iron, 1.0% calcium, 0.7% phosphorus). Fgf23 eGFP/eGFP homozygotes were not generated due to known early growth retardation and mortality in Fgf23 null mice. After blood and tissue collection at 8 weeks of age, phenotypes of the 4 Hbb-Fgf23 genotype combinations were compared by two-way ANOVA. Compared to mice with intact Hbb and Fgf23 alleles ( Hbb +/+ Fgf23 +/+), Hbb th3/+ Fgf23 +/+ mice showed markedly elevated plasma levels of “total” FGF23 (sum of intact, active hormone and C-terminal cleaved fragments). Plasma total FGF23 levels in Hbb th3/+ Fgf23 +/+ and Hbb th3/+ Fgf23 +/eGFP mice were similar, indicating utility of the latter genotype for interrogating sites of increased Fgf23 expression in murine b-thalassemia. We therefore used anti-GFP immunohistochemistry in formalin-fixed bone marrow sections to assess Fgf23 eGFP reporter allele expression in the context of tissue architecture. Hbb th3/+ Fgf23 +/eGFP mice showed GFP expression in BM-SEC throughout the bone marrow, which was more intense than in non-anemic Hbb +/+ Fgf23 +/eGFP mice; controls lacking the Fgf23 eGFP allele confirmed anti-GFP antibody specificity. Hbb th3/+ mice also showed Fgf23 eGFP reporter expression in the thymic vasculature but not vasculature of the liver, spleen, or kidney. In contrast to their marked elevation in plasma total FGF23, Hbb th3/+ Fgf23 +/+ mice showed plasma levels of the intact, biologically-active FGF23 hormone that overlapped with levels in Hbb +/+ Fgf23 +/+ controls. Interestingly, compared with Hbb th3/+ Fgf23 +/+ mice, Hbb th3/+ Fgf23 +/eGFP mice showed 45% lower plasma intact FGF23, suggesting an effect of Fgf23 gene dosage. Given prior reported effects of FGF23 on hematopoiesis, we assessed if Fgf23 gene dosage modulated the severity of ineffective erythropoiesis in Hbb th3/+ mice. Compared to Hbb +/+ Fgf23 +/+ controls, Hbb th3/+ Fgf23 +/+ mice showed reductions in RBC, hemoglobin, and mean corpuscular hemoglobin, as well as increases in RDW and in spleen weight (reflecting extramedullary hematopoiesis). However, the severity of these parameters was not modified by heterozygous Fgf23 disruption ( Hbb th3/+ Fgf23 +/eGFP).While FGF23 has been reported to induce left ventricular hypertrophy [PMID:21985788], Hbb th3/+ Fgf23 +/+ and Hbb th3/+ Fgf23 +/eGFP mice showed similarly increased heart-to-body weight ratios compared to Hbb +/+ Fgf23 +/+ controls. In non-thalassemic mice, heterozygous Fgf23 disruption ( Hbb +/+ Fgf23 +/eGFP) caused a trend ( P=0.10) towards lower plasma intact FGF23 while not impacting hematological parameters or spleen weight. In summary, by showing that Fgf23 gene dosage modulates plasma intact FGF23 levels in Hbb th3/+ mice without altering severity of anemia or splenomegaly, our data suggest that moderate reductions in circulating levels of the intact FGF23 hormone do not alter the severity of ineffective erythropoiesis in Hbb th3/+mice at 8 weeks of age. Our finding that β-thalassemic mice exhibit Fgf23 upregulation in BM-SEC lays the groundwork for future investigation of the functional effects of locally-produced FGF23 in the thalassemic hematopoietic stem cell niche.

Bone Marrow Sinusoidal Endothelial Cells Are a Site of Fgf23 Upregulation in Iron Deficiency Anemia

Iron deficiency anemia (IDA) has been identified as a potent stimulator of FGF23 (fibroblast growth factor 23), a phosphaturic hormone classically thought to be produced by bone-embedded osteocytes. Recently, both phlebotomy and erythropoietin administration have been shown to upregulate FGF23 production in bone marrow. However, the cell type(s) mediating FGF23 upregulation in states of perturbed erythropoiesis require further clarification.Tmprss6-/- mice exhibit hepcidin elevation leading to systemic iron deficiency and iron-restricted anemia. We previously reported that Tmprss6-/- mice exhibit altered phosphate balance, elevated circulating FGF23, and Fgf23 mRNA upregulation in bone marrow but not cortical bone. Here, we clarify the sites of Fgf23 promoter activity in Tmprss6 -/- bone marrow using a reporter allele in which the enhanced green fluorescent protein (eGFP) coding sequence has been knocked into the endogenous Fgf23 locus. We generated Tmprss6 +/+,Tmprss6 +/-, and Tmprss6 -/- littermates of both sexes that carried either one (Fgf23 +/eGFP) or zero (Fgf23 +/+) copies of the reporter allele. Tmprss6-/- mice showed hyperhepcidinemia, hypoferremia, microcytic anemia, and tissue iron deficiency, which were not altered by heterozygous Fgf23 disruption (Figure 1A-C). By ELISA, Tmprss6-/- Fgf23 +/eGFP mice showed plasma levels of “total” FGF23 (intact, active hormone and C-terminal cleaved fragments) that remained markedly elevated compared to Tmprss6+/+ littermates (Figure 1D). Total FGF23 elevation in Tmprss6-/- Fgf23 +/eGFP mice was slightly less pronounced than Tmprss6-/- Fgf23 +/+ mice, suggesting an effect of Fgf23 gene dosage. In mice with 2 intact Fgf23 alleles, serum erythropoietin showed a strong linear correlation with plasma total FGF23.By confocal imaging, femurs of mice carrying the Fgf23 eGFP allele showed green fluorescence in vascular regions of the bone marrow but not in the bone cortex. Green fluorescence was more intense in Tmprss6-/- Fgf23+/eGFP mice than non-anemic controls. By flow cytometry of enzymatically digested bone marrow, we observed bright green fluorescence in a subset of endothelial cells (CD45 - Ter119 - CD31 +) exclusively in mice carrying the Fgf23 eGFP reporter allele (Figure 1E). The percentage of endothelial cells that were GFP bright was higher in Tmprss6-/- Fgf23 +/eGFP versus non-anemic mice. To clarify the endothelial cell subtype that expresses Fgf23, we mined published transcriptomic datasets from mice of normal iron balance and discovered higher Fgf23 mRNA in bone marrow sinusoidal endothelial cells compared to other bone marrow endothelial cell populations. Accordingly, we used anti-GFP immunohistochemistry in formalin-fixed bone marrow sections to assess Fgf23 eGFP reporter allele expression in the context of tissue architecture. Tmprss6-/- Fgf23 +/eGFP mice showed GFP expression in bone marrow sinusoidal endothelial cells, which was more intense than in non-anemic controls (Figure 1F). GFP reporter expression was also detected in rare cells of the thymus but not in liver, spleen, heart, muscle, or kidney. Collectively, our data reveal that bone marrow sinusoidal endothelial cells are a site of Fgf23 upregulation in chronic IDA.Because IDA in Tmprss6-/- mice results from pathologic hepcidin elevation, we also sought to determine if bone marrow sinusoidal endothelial cells are a site of Fgf23 upregulation in anemic mice with intact hepcidin regulation. We therefore subjected Fgf23 +/eGFP mice (with 2 intact Tmprss6 alleles) to a 500µl phlebotomy regimen (with saline volume replacement) known to induce marked anemia and hepcidin suppression. Compared to non-phlebotomized Fgf23 +/eGFP controls, phlebotomized Fgf23 +/eGFP mice showed severe anemia, elevated serum erythropoietin, and elevated plasma FGF23 18 hours after blood loss. Additionally, immunohistochemistry revealed more intense GFP expression in bone marrow sinusoidal endothelial cells of phlebotomized Fgf23 +/eGFP mice than non-phlebotomized controls. Taken together, our results show for the first time that bone marrow sinusoidal endothelial cells are a site of Fgf23 upregulation in both acute and chronic anemia. Given the serum erythropoietin elevation in both models, our findings suggest that erythropoietin may act directly or indirectly on sinusoidal endothelial cells to promote FGF23 production during anemia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

β-Galactosylceramidase Deficiency Causes Bone Marrow Vascular Defects in an Animal Model of Krabbe Disease.

Krabbe disease (KD) is an autosomal recessive sphingolipidosis caused by the deficiency of the lysosomal hydrolase β-galactosylceramidase (GALC). Oligodendroglia degeneration and demyelination of the nervous system lead to neurological dysfunctions which are usually lethal by two years of age. At present, the only clinical treatment with any proven efficacy is hematopoietic stem-cell transplantation, which is more effective when administered in the neonatal period to presymptomatic recipients. Bone marrow (BM) sinusoidal endothelial cells (SECs) play a pivotal role in stem cell engraftment and reconstitution of hematopoiesis. Previous observations had shown significant alterations of microvascular endothelial cells in the brain of KD patients and in Galc mutant twitcher mice, an authentic model of the disease. In the present study, we investigated the vascular component of the BM in the femurs of symptomatic homozygous twitcher mice at postnatal day P36. Histological, immunohistochemical, and two-photon microscopy imaging analyses revealed the presence of significant alterations of the diaphyseal BM vasculature, characterized by enlarged, discontinuous, and hemorrhagic SECs that express the endothelial marker vascular endothelial growth factor receptor-2 (VEGFR2) but lack platelet/endothelial cell adhesion molecule-1 (CD31) expression. In addition, computer-aided image analysis indicates that twitcher CD31−/VEGFR2+ SECs show a significant increase in lumen size and in the number and size of endothelial gaps compared to BM SECs of wild type littermates. These results suggest that morphofunctional defects in the BM vascular niche may contribute to the limited therapeutic efficacy of hematopoietic stem-cell transplantation in KD patients at symptomatic stages of the disease.

2021 - CHOLINERGIC INFLUENCE OF BONE MARROW VASCULAR MICROENVIRONMENT INFLUENCES HEMATOPOIETIC STEM CELL MOBILIZATION

The regulation of HSC movement between the bone marrow (BM) microenvironment and the circulation remains an important clinical target in the setting of BM transplantation. The influence of parts of the nervous system, the sympathetic arm in particular, on BM niche function and mobilization have been investigated. However, the basal role of the cholinergic system in regulating HSC mobilisation is yet to be elucidated. We show that the cholinergic system and specifically the nicotinic -7 acetylcholine receptor (Chrna7) mediates alterations in the structure and function of the BM sinusoidal endothelial cells as well as perivascular stromal cells (LepR+). Inhibition of this receptor with MLA leads to a loss of integrity in the BM sinusoidal structures along with a loss in perivascular niche stem cell factor production. Blocking Chrna7 leads to mobilisation of functional HSCs without skewing that is typically present in G-CSF mobilization. This is suggested by single cell RNA sequencing of MLA mobilised peripheral HSCs that indicates a more balanced genetic lineage signature compared to those mobilised with G-CSF. In addition, administering MLA during G-CSF treatment leads to a synergetic effect, while stimulation of the Chrna7 receptor with GTS-21 blunts G-CSF induced HSC mobilisation. These findings suggest a novel role for the peripheral cholinergic system in HSC mobilisation via niche cell alterations that provides a G-CSF independent alternative route for BM transplantation.

Bone marrow sinusoidal endothelium as a facilitator/regulator of cell egress from the bone marrow.

Despite more attention to cell migration from circulation into the bone marrow (BM), particularly homing of haematopoietic stem/progenitor cells, the process and mechanisms of cell mobilisation from the BM into the circulation remain largely underexplored. The process of cell mobilisation or transcellular cell migration from BM into the circulation (cell egress) is a crucial biological process in mammals as it is important to maintain homeostasis of various physiological functions including, but not limited to, the immune system, erythropoiesis, platelet release, and stem cell migration. The BM microvascular system composes of a monolayer of specialized endothelial cells, called sinusoidal endothelial cells (SECs). While it is very well evident that the process of cell egress occurs exclusively through BM SECs, there is a lack of systematic analyses addressing the extent of contribution of BM SECs to the process of cell egress from the BM. Therefore, this review aims to address the potential ability of BM SECs in regulating and/or facilitating the process of cell egress from BM. In this review, we address, firstly, the unique ultra-/structural and molecular features of BM SECs and discuss the possible biological interactions between BM SECs and various egressing cells in physiological conditions. Secondly, we propose the potential role of BM SECs in egress of leukemic cells from BM into the circulation. Finally, we discuss the potential role of BM SECs in homing of haematopoietic stem cells. Collectively, the current review suggests that the BM SECs may not be merely a neutral gatekeeper for cell intravasation and extravasation, but rather is a dynamic trafficking surveillance system, thereby the process of BM cell egress/mobilisation can be regulated.

Flavonoid genistein protects bone marrow sinusoidal blood vessels from damage by methotrexate therapy in rats.

Cancer chemotherapy can cause significant damage to the bone marrow (BM) microvascular (sinusoidal) system. Investigations must now address whether and how BM sinusoidal endothelial cells (SECs) can be protected during chemotherapy. Herein we examined the potential protective effects of genistein, a soy-derived flavonoid, against BM sinusoidal damage caused by treatment with methotrexate (MTX). The groups of young adult rats were gavaged daily with genistein (20 mg/kg) or placebo. After 1 week, rats also received daily injections of MTX (0.75 mg/kg) or saline for 5 days and were killed after a further 4 days. Histological analyses showed that BM sinusoids were markedly dilated ( p < 0.001) in the MTX-alone group but were unaffected or less dilated in the genistein+MTX group. In control rats, genistein significantly enhanced expression of vascular endothelial growth factor (VEGF; p < 0.01), particularly in osteoblasts, and angiogenesis marker CD31 ( p < 0.001) in bone. In MTX-treated rats, genistein suppressed MTX-induced apoptosis of BM SECs ( p < 0.001 vs MTX alone group) and tended to increase expression of CD31 and VEGF ( p < 0.05). Our in vitro studies showed that genistein in certain concentrations protected cultured SECs from MTX cytotoxic effects. Genistein enhanced tube formation of cultured SECs, which is associated with its ability to induce expression of endothelial nitric oxide synthase and production of nitric oxide. These data suggest that genistein can protect BM sinusoids during MTX therapy, which is associated, at least partially, with its indirect effect of promoting VEGF expression in osteoblasts and its direct effect of enhancing nitric oxide production in SECs.

Methotrexate chemotherapy–induced damages in bone marrow sinusoids: An in vivo and in vitro study

AbstractChemotherapeutic agents are very well evident extrinsic stimuli for causing damage to endothelial cells. Methotrexate is an antimetabolite commonly used to treat solid tumours and paediatric cancers. However, studies on the effect(s) of methotrexate on bone marrow microvascular system are inadequate. In the current study, we observed a significant bone marrow microvascular dilation following methotrexate therapy in rats, accompanied by apoptosis induction in bone marrow sinusoidal endothelial cells, and followed by recovery of bone marrow sinusoids associated with increased proliferation of remaining bone marrow sinusoidal endothelial cells. Our in vitro studies revealed that methotrexate is cytotoxic for cultured sinusoidal endothelial cells and can also induce apoptosis which is associated with upregulation of expression ratio of Bax and Bcl‐2 genes and Bax/Bcl‐2 expression ratio. Furthermore, it was shown that methotrexate can negatively affect proliferation of cultured sinusoidal endothelial cells and also inhibit their abilities of migration and formation of microvessel like tubes. The data from this study indicates that methotrexate can cause significant bone marrow sinusoidal endothelium damage in vivo and induce apoptosis and inhibit proliferation, migration and tube‐forming abilities of sinusoidal endothelial cells in vitro.

Isolation of murine bone marrow scavenging sinusoidal endothelial cells.

The bone marrow (BM) is permeated with sinusoidal vessels lined with sinusoidal endothelial cells (SEC), which are crucial for BM physiology and hemopoietic stem cell (HSC) renewal. However, little is known about the characteristics or functional capacity of bone marrow sinusoidal endothelial cells (BMSEC). One significant barrier to the study of BMSEC is the lack of specific cell surface markers that can be used to isolate these cells. Nevertheless, BMSEC possess one exceptional property, namely, the ability to scavenge large amounts of soluble waste molecules such as advanced glycation end-products (AGE) and we have utilized this to label BMSEC for cell sorting and isolation. We describe the means to produce and fluorescently label AGE, its use as a functional in vivo marker of BMSEC and the isolation of these cells from murine BM using fluorescent activated cell separation (FACS).

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 (CD45neg 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 (NPY3-36) 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. Disclosures:Hoggatt:Fate Therapeutics: Consultancy. Pelus:Fate Therapeutics: Consultancy.

Sinusoidal Endothelial CD26 Cleavage of Neuropeptide Y Regulates Transendothelial Migration and Mobilization of Hematopoietic Stem and Progenitor Cells in Response to G-CSF.

Abstract 2347Hematopoietic stem and progenitor cell (HSPC) mobilization by G-CSF is reduced in CD26 knockout (−/−) mice or mice treated with compounds that inhibit CD26 enzyme activity. However, how CD26 regulates mobilization is not known. CD26 is a surface dipeptidyl peptidase expressed on hematopoietic and non-hematopoietic cells. The fact that CD26 is expressed on HSPC, has suggested that intrinsic HSPC CD26 is involved in their mobilization. We reasoned that if intrinsic HSPC CD26 is crucial for their mobilization, then CD26+ HSPC would mobilize more readily than CD26− HSPC. After G-CSF treatment, CD26+ SLAM LSK and LSK cells increased 2 and 3.7-fold in peripheral blood (PB) whereas CD26− SLAM LSK and LSK cells increased 3.9 and 8.7 fold respectively; with the ratio of CD26+/CD26− HSPC in the bone marrow (BM) remaining unchanged. In chimeric mice where hematopoietic cells were either wild-type or CD26−/− in origin and the stromal environment was wild-type, equivalent CFU-GM and LSK cells were mobilized by G-CSF. These studies suggest that CD26 expression on hematopoietic origin cells including HSPC is not required for the G-CSF induced mobilization of HSPC.The BM stromal environment includes both osteolineage cells and endothelial cells (EC). To explore whether osteolineage cell CD26 is involved in G-CSF mobilization, we quantitated CD45−CD119−CD31−Sca-1+ Alcam− cells enriched for mesenchymal progenitor cells (MPC) and CD45−CD119−CD31− Sca-1- Alcam+ cells enriched for osteoblasts (OB) in the BM of mice treated with G-CSF or G-CSF plus the CD26 inhibitor diprotin A. Total MPC and OB number as well as the retention factors SDF-1, SCF-1 and osteoprotegerin (OPG) were reduced equally in the BM of G-CSF and G-CSF plus diprotin A treated mice, suggesting that osteolineage cell CD26 is not involved in mobilization. Since endothelial cells (EC) express CD26 and HSPC resides proximal to sinusoidal endothelial cells (SEC), we evaluated if SEC CD26 has a role in G-CSF induced HSPC mobilization. G-CSF treatment increased CD26 expression on CD45- Ter119− Sca-1− VEGFR3+ SEC by 2-fold. Because mobilized HSPC must transmigrate across SEC to enter the PB, we determined if EC CD26 regulates HSPC transmigration. Treatment of confluent EC monolayers with G-CSF in vitro, increased transendothelial migration of both wild-type and CD26−/−HSPC, which was significantly reduced by diprotin A, supporting the hypothesis that enhanced expression of CD26 on BM SEC by G-CSF is involved in HSPC mobilization.Since SDF-1 is a BM retention factor for HSPC and CD26 can cleave SDF-1 in vitro, it is possible that SDF-1 degradation by CD26 plays a role in HSPC egress. However, direct evidence supporting proteolytic inactivation of SDF-1 by CD26 in response to G-CSF is not available. Using a novel bio-binding assay and mass spectroscopy, we found similar levels of active SDF-1 in the BM extracellular fluid and on SEC membranes from G-CSF treated wild-type and CD26−/− mice, suggesting that G-CSF mobilization is not linked to SDF-1degradation. We therefore searched protein sequence databases for known EC products containing a CD26 recognition site and implicated in HSPC trafficking. We found that the neurotransmitter neuropeptide Y (NPY), a sympathetic nerve and EC product contains an N-terminal Tyr-Pro sequence and can be cleaved to NPY3–36 by CD26. We hypothesized that if cleavage of NPY by EC CD26 is involved in transendothelial migration of HSPC, then reduced HSPC migration through EC monolayers in the presence of G-CSF plus diprotin A would be reversed in the presence of NPY3–36. As expected, NPY3–36 reversed the diprotin A mediated attenuation in HSPC migration through G-CSF treated EC. In vivo, the attenuated response to G-CSF mobilization in diprotin A treated or CD26−/− mice could be completely reversed by co-administration of NPY3–36, but not full length NPY. In G-CSF mobilized wild-type mice, we found that expression of gap junction proteins on BM SEC, including CD31 and VE-cadherin are reduced coincident with increased EC permeability, which is attenuated by co-administration of diptotin A and restored to normal in mice receiving G-CSF plus diprotin A and NPY3–36, but not full length NPY. In conclusion, we have found that CD26 regulates G-CSF induced mobilization that is independent of the SDF-1 paradigm; rather elevated SEC CD26 in response to G-CSF enhances transendothelial migration and mobilization of HSPC via cleavage of NPY. Disclosures:No relevant conflicts of interest to declare.

Pleiotrophin Regulates the Retention and Self-Renewal of Hematopoietic Stem Cells in the Bone Marrow Vascular Niche

The mechanisms through which the bone marrow (BM) microenvironment regulates hematopoietic stem cell (HSC) fate remain incompletely understood. We examined the role of the heparin-binding growth factor pleiotrophin (PTN) in regulating HSC function in the niche. PTN(-/-) mice displayed significantly decreased BM HSC content and impaired hematopoietic regeneration following myelosuppression. Conversely, mice lacking protein tyrosine phosphatase receptor zeta, which is inactivated by PTN, displayed significantly increased BM HSC content. Transplant studies revealed that PTN action was not HSC autonomous, but rather was mediated by the BM microenvironment. Interestingly, PTN was differentially expressed and secreted by BM sinusoidal endothelial cells within the vascular niche. Furthermore, systemic administration of anti-PTN antibody in mice substantially impaired both the homing of hematopoietic progenitor cells to the niche and the retention of BM HSCs in the niche. PTN is a secreted component of the BM vascular niche that regulates HSC self-renewal and retention invivo.

Angiopoietin-like protein 3 supports the activity of hematopoietic stem cells in the bone marrow niche

AbstractThe physiologic roles of angiopoietin-like proteins (Angptls) in the hematopoietic system remain unknown. Here we show that hematopoietic stem cells (HSCs) in Angptl3-null mice are decreased in number and quiescence. HSCs transplanted into Angptl3-null recipient mice exhibited impaired repopulation. Bone marrow sinusoidal endothelial cells express high levels of Angptl3 and are adjacent to HSCs. Importantly, bone marrow stromal cells or endothelium deficient in Angptl3 have a significantly decreased ability to support the expansion of repopulating HSCs. Angptl3 represses the expression of the transcription factor Ikaros, whose unregulated overexpression diminishes the repopulation activity of HSCs. Angptl3, as an extrinsic factor, thus supports the stemness of HSCs in the bone marrow niche.

Stabilins are expressed in bone marrow sinusoidal endothelial cells and mediate scavenging and cell adhesive functions

Bone marrow sinusoidal endothelial cells have a specific function as a site of transmigration of hematopoietic stem and progenitor cells and mature blood cells between bone marrow and blood stream. However, the specific characteristics of bone marrow sinusoidal endothelial cells are still largely unclear. We here report that these cells express stabilin-1 and stabilin-2, which in liver sinusoidal endothelial cells have been identified as endocytic scavenger receptors for several ligands, including SPARC and hyaluronan. We show here that intravenously injected formaldehyde-treated serum albumin, advanced glycation end-products, and collagen I α-chains were taken up by bone marrow sinusoidal endothelial cells, showing that these cells have a scavenging function and thereby may modulate bone marrow vascular stem cell niches. Importantly, we show hyaluronan mediated adhesion of hematopoietic stem and progenitor cells to stabilin-2-transfected cells, suggesting that stabilin-2 contributes to adhesion and homing of circulating stem and progenitor cells to bone marrow.

Stabilin-1 and Stabilin-2 Are Expressed in Bone Marrow Sinusoidal Endothelial Cells and Mediate Scavenging and Cell Adhesive Functions

Stabilin-1 and stabilin-2 have been identified as scavenging receptors in sinusoidal capillaries in some tissues, including liver and spleen. They function as endocytic receptors for SPARC and hyaluronan, respectively, and for several other ligands. In the present study, we show by real-time PCR, Western blot and immunohistochemistry that bone marrow (BM) sinusoidal endothelial cells (SECs) ubiquitously express stabilin-1 and stabilin-2. To test the ability of BM SECs to function as a scavenging endothelium, we analyzed the uptake of specific scavenger receptor ligands after intravenous injection. Accumulation of TRITC labelled formaldehyde-treated serum albumin (FSA) was observed one hour after the injection in the BM SECs. Injection of unlabelled FSA together with TRITC-FSA reduced the fluorescence in the SECs, indicating that the uptake was due to specific recognition of FSA. Likewise, FITC-conjugated advanced glycation end products (AGEs)-modified BSA accumulated in BM SECs. These two ligands are primarily cleared by the stabilins. These results suggested that bone marrow SECs have a scavenging function. Stabilin-2 has been identified as a major receptor for hyaluronan. Hyaluronan is synthesized by primitive hematopoietic cells and has been shown to influence stem and progenitor (HSPC) functions, including mobilization and homing into BM (Nilsson et al., Blood. 2003; 101:856). Consequently, it is possible that adhesion of hyaluronan on HSPCs to stabilin-2 is a recognition mechanism in BM SECs, directing circulating HSPCs into BM. To investigate this, we studied adhesion of mouse BM lin-Sca-1+Kit+ (LSK) HSPCs to stabilin-1 or stabilin-2 transfected human embryonic kidney cells (HEK 293 cells). We found increased adhesion of LSK cells to stabilin-2 expressing cells, as compared to stabilin-1 expressing or non-transfected cells. Notably, hyaluronidase treatment abolished the increased adhesion of the LSK cell to stabilin-2 transfected cells. These findings indicate that stabilin-2 mediates adhesion of HSPCs to bone marrow SECs. In conclusion, this study shows a novel function for BM SECs as a scavenging endothelium expressing stabilin-1 and stabilin-2. The specific function of stabilins in recognition and endocytosis of extracellular matrix molecules, including hyaluronan and SPARC (Kzhyshkowska J et al., J Cell Mol Med. 2006; 10:635), suggests that these receptors are involved in tissue remodelling and cell trafficking in BM. Importantly, hyaluronan-mediated binding of HSPCs to SEC stabilin-2 may be a specific recognition mechanism for HSPCs in BM sinusoids. Similarly, binding of tumour cell-associated hyaluronan by stabilin-2 might cause tumour cells in blood to halt in bone marrow, thereby increasing the likelihood for metastasis at this site.

Bone marrow sinusoidal endothelial cells undergo nonapoptotic cell death and are replaced by proliferating sinusoidal cells in situ to maintain the vascular niche following lethal irradiation

Bone marrow sinusoids remain predominantly host-derived following bone marrow transplantation. Systematic analysis was conducted at the cellular level to investigate how the host sinusoidal structures survived after lethal irradiation. Apoptosis and cell proliferation assays were performed on bone marrow sections at various time points during the first 2 weeks postirradiation to study the extent of damage to sinusoidal endothelial cells from lethal irradiation and to determine whether cell proliferation contributes to the recovery of the sinusoidal system. Phosphorylated H2AX was present in both hematopoietic and sinusoidal endothelial cells 3 hours after irradiation demonstrating DNA damage. Three days after irradiation, some sinusoidal endothelial cells became terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling -positive, but were caspase-3 and in situ oligo ligation -negative, suggesting nonapoptotic DNA fragmentation. Clusters of sinusoidal endothelial cells that expressed Ki67 appeared 3 days after irradiation, and increased through day 7. These Ki67-positive endothelial cells were host-derived. Bromodeoxyuridine-positive endothelial cells were present in the Ki67-positive areas confirming endothelial cell replication. Twenty percent of the sinusoidal endothelial cells were lost by day 3 after irradiation. The total number of endothelial cells remained relatively unchanged between day 3 and day 14. These results demonstrate that lethal irradiation resulted in limited, nonapoptotic sinusoidal endothelial cell loss, followed by proliferation of preexisting host-derived mature sinusoidal endothelial cells. Our data suggest that DNA repair mechanisms and proliferation of host endothelial cells within the sinusoids are involved in maintenance of the structural integrity of the bone marrow vascular niche following lethal irradiation.

Culturing of Human Mesenchymal Stem Cells as Three-dimensional Aggregates Induces Functional Expression of CXCR4 That Regulates Adhesion to Endothelial Cells

Culture-expanded human mesenchymal stem cells (hMSCs) are increasingly used in a variety of preclinical and clinical studies. However, these cells have a low rate of engraftment to bone marrow or damaged tissues. Several laboratories have shown that during isolation and subculturing mesenchymal stem cells quickly lose the expression of CXCR4, the key receptor responsible for lymphocytes and hematopoietic stem cell homing. Here we show that culturing of hMSCs as three-dimensional aggregates (hMSC spheroids) restores CXCR4 functional expression. Expression of CXCR4 inversely correlates with the secretion of SDF-1 by hMSCs. Cells from hMSC spheroids up-regulate expression of CD49b, the alpha2 integrin subunit, and suppress the expression of CD49d, the alpha4 integrin subunit. Transfer of cells from the spheroids back to a monolayer suppresses the expression of CXCR4 and CD49b and restores the expression of CD49d. Treatment of cells from the spheroids with SDF-1 leads to CXCR4 internalization and activation of ERK-1,2. Adhesion of hMSCs to human umbilical vein endothelial cells (HUVECs) was investigated. SDF-1, AMD-3100, or exposure of HUVECs to hypoxia did not affect adhesion of hMSCs from a monolayer to HUVECs. Adhesion of cells from hMSC spheroids to HUVECs was stimulated by SDF-1, AMD-3100, or by exposure of HUVECs to hypoxia. Stimulatory effects of hypoxia and addition of SDF-1 or AMD-3100 were not additive. Overall, our data indicate that the expression of CXCR4 by hMSCs regulates hMSC adhesion to endothelial cells.

Interactions of LSECtin and DC-SIGN/DC-SIGNR with viral ligands: Differential pH dependence, internalization and virion binding

The calcium-dependent lectins DC-SIGN and DC-SIGNR (collectively termed DC-SIGN/R) bind to high-mannose carbohydrates on a variety of viruses. In contrast, the related lectin LSECtin does not recognize mannose-rich glycans and interacts with a more restricted spectrum of viruses. Here, we analyzed whether these lectins differ in their mode of ligand engagement. LSECtin and DC-SIGNR, which we found to be co-expressed by liver, lymph node and bone marrow sinusoidal endothelial cells, bound to soluble Ebola virus glycoprotein (EBOV-GP) with comparable affinities. Similarly, LSECtin, DC-SIGN and the Langerhans cell-specific lectin Langerin readily bound to soluble human immunodeficiency virus type-1 (HIV-1) GP. However, only DC-SIGN captured HIV-1 particles, indicating that binding to soluble GP is not necessarily predictive of binding to virion-associated GP. Capture of EBOV-GP by LSECtin triggered ligand internalization, suggesting that LSECtin like DC-SIGN might function as an antigen uptake receptor. However, the intracellular fate of lectin–ligand complexes might differ. Thus, exposure to low-pH medium, which mimics the acidic luminal environment in endosomes/lysosomes, released ligand bound to DC-SIGN/R but had no effect on LSECtin interactions with ligand. Our results reveal important differences between pathogen capture by DC-SIGN/R and LSECtin and hint towards different biological functions of these lectins.

Identification of Pleiotrophin as a Novel Growth Factor for Hematopoietic Stem and Progenitor Cells.

Hematopoietic stem and progenitor cells have been demonstrated to reside in association with bone marrow sinusoidal endothelial cells in vivo but the mechanisms through which the BM vascular niche regulates hematopoiesis remains incompletely defined. In an attempt to identify candidate soluble growth factors for hematopoietic stem cells (HSCs) which are produced by endothelial cells (Ecs), we have performed comparative genome-wide expression analysis of a primary human brain-derived Ecs which we have previously demonstrated to support a 10-fold expansion of human SCID-repopulating cells (SRCs) in non-contact cultures. This analysis revealed that pleiotrophin (PTN), a heparin binding growth factor, was 32-fold overexpressed in HUBECs that supported human HSC expansion as compared to non-supportive EC lines. Pleiotrophin has anti-apoptotic, mitogenic and transforming activities, suggesting a role in tumorigenesis. No role for pleiotrophin in hematopoiesis has been described. In order to determine if PTN contributes to the demonstrated HSC-supportive activity of HUBECs, the activity of PTN was blocked by adminstration of a neutralizing anti-PTN mAb to the coculture model. Three doses of PTN neutralizing antibody (5, 25, and 50 ug/ml) were added to non-contact cocultures of HUBECs with human cord blood CD34+CD38-lin- HSCs. The anti-PTN treatment caused a decrease in the numbers of phenotypic HSCs in culture in a dose dependent manner. Relative to control HUBEC cultures, the % and expansion of CD34(+) progenitors decreased signficantly at the two higher doses of anti-PTN (p<0.05, respectively). Additionally, CD34(+)CD38(−) lin(−) progenitors were decreased by 33% at the highest dose of anti-PTN compared to control HUBEC cultures (p<0.05). Transplantation studies of anti-PTN treated versus control HUBEC-cultured progeny into NOD/SCID mice are ongoing to confirm the central role of PTN in the expansion of human HSCs in culture. In a correlative study, the addition of recombinant PTN to cultures of human HSCs with thrombopoietin, SCF and flt-3 ligand was shown to significantly increase CD34+ cell content and CD34+CD38- cell content compared to cytokines alone, again in a dose-dependent manner. Taken together these results suggest that PTN, a growth factor with no previously ascribed hematopoietic activity, is a soluble growth factor for human hematopoietic progenitor cells. Since blockade of PTN activity significantly inhibits the soluble activity of HUBECs to support human HSC proliferation and expansion, PTN is also a candidate growth factor for further study in the context of understanding the function of the BM vascular niche.

Endothelial Progenitor Cell Transplantation for Hematopoietic Regeneration.

Hematopoietic stem cells (HSCs) reside in close association with bone marrow (BM) osteoblasts which help maintain the quiescent HSC pool over time. Hematopoietic stem and progenitor cells also reside in association with BM sinusoidal endothelial cells in vivo but the function of the BM vascular niche in regulating hematopoiesis is less well defined. We hypothesized that the BM endothelial cells function in vivo to support the proliferation and regeneration of hematopoietic stem/progenitor cells and that damage incurred by the BM vascular niche following myelotoxic therapies contributes to delayed hematopoietic recovery in vivo. To test this hypothesis, we generated primary endothelial progenitor cells (EPCs) from the peripheral blood of C57Bl6 mice and examined whether transplantation of allogeneic EPCs alone could facilitate earlier hematopoietic recovery in irradiated recipient BALB/c mice as compared to controls. Ten week old BALB/c mice were irradiated with 550 cGy total body irradiation at day 0 and then received either 1 x 10e6 EPCs intravenously at +4 hours and 2.5 x 10e6 EPCs daily intraperitoneally on days +1 to +4 or saline infusions at the same intervals. Microscopic analysis of femurs from BALB/c mice demonstrated significantly reduced BM cellularity at day +10 and +15, with recovery evident at day +20. EPC-transplanted mice also showed marked BM hypocellularity at day +10 but earlier recovery of BM cellularity at day +15. Total viable BM cell counts confirmed a 2-fold increase in viable BM cells in the EPC-transplanted group at day +15 compared to controls. Flow cytometric analysis revealed that EPC-transplanted mice had significantly earlier recovery of c-kit+sca-1-lin− progenitor cells at day +10, +12 and +15 compared to saline treated controls (means 0.9%, 0.2%, 1.3% vs. 0.2%, 0.06%, 0.5%; p=0.08, p=0.02, p=0.04, respectively). EPC-transplanted mice also demonstrated accelerated regeneration of c-kit+sca-1+lin− (“KSL”) stem/progenitor cells at day +15 compared to saline-treated controls (mean 0.09% vs. 0.03% KSL cells, p=0.04). Importantly, this accelerated recovery of hematopoiesis corresponded with more rapid recovery of hematologic parameters in EPC-transplanted mice compared to controls. EPC-transplanted mice demonstrated significantly earlier recovery of total white blood cells compared to controls by day +16 (mean 1.4 vs. 0.5, p<0.01) and achieved recovery of absolute neutrophil count (ANC) > 500 at day +18; conversely, controls did not achieve ANC > 500 until day +30 (p<0.01 for all timepoints, day +10 to +21). Platelet count recovery was also accelerated in EPC-transplanted mice with a platelet count of >400,000 achieved at day +14 versus day +25 in the saline-treated controls (p<0.01 for all timepoints, day +10 to +18). Preliminary studies using GFP+ endothelial cells have shown no evidence of engraftment of transplanted endothelial cells in the BM or spleen of recipient mice, suggesting that an EPC-elaborated soluble activity may account for the results observed. In ongoing studies, we are examining whether transplanted EPCs mediate these effects indirectly via accelerated recovery of MECA+ endothelial cells in the BM vascular niche or directly upon hematopoietic stem/progenitor cells. Taken together, these studies demonstrate that transplantation of primary EPCs alone mediates accelerated hematopoietic recovery following myelotoxic therapy and these effects are not impeded by immunologic barriers. These results also confirm the contribution of endothelial cells in supporting hematopoietic regeneration in vivo.