Search

Aging associated defects within stem cell-supportive niches contribute towards age-related decline in stem cell activity. However, mechanisms underlying age-related niche defects, and whether restoring niche function can improve stem cell fitness, remain unclear. Here, we sought to determine whether aged blood stem cell function can be restored by rejuvenating their supportive niches within the bone marrow (BM). We identify Netrin-1 as a critical regulator of BM niche cell aging. Niche-specific deletion of Netrin-1 induces premature aging phenotypes within the BM microenvironment, while supplementation of aged mice with Netrin-1 rejuvenates aged niche cells and restores competitive fitness of aged blood stem cells to youthful levels. We show that Netrin-1 plays an essential role in maintaining active DNA damage responses (DDR), and that aging-associated decline in niche-derived Netrin-1 results in DNA damage accumulation within the BM microenvironment. We show that Netrin-1 supplementation is sufficient to resolve DNA damage and restore regenerative potential of the aged BM niche and blood stem cells to endure serial chemotherapy regimens., (© 2023. The Author(s).)

In this issue of Cancer Cell, Passaro et al. demonstrate how leukemia through aberrant induction of reactive oxygen species and nitric oxide production trigger marrow vessel leakiness, instigating pro-leukemic function. Disrupted tumor blood vessels promote exhaustion of non-malignant stem and progenitor cells and may facilitate leukemia relapse following chemotherapeutic treatment., (Copyright © 2017. Published by Elsevier Inc.)

Bone marrow endothelial cells (BMECs) form a network of blood vessels that regulate both leukocyte trafficking and haematopoietic stem and progenitor cell (HSPC) maintenance. However, it is not clear how BMECs balance these dual roles, and whether these events occur at the same vascular site. We found that mammalian bone marrow stem cell maintenance and leukocyte trafficking are regulated by distinct blood vessel types with different permeability properties. Less permeable arterial blood vessels maintain haematopoietic stem cells in a low reactive oxygen species (ROS) state, whereas the more permeable sinusoids promote HSPC activation and are the exclusive site for immature and mature leukocyte trafficking to and from the bone marrow. A functional consequence of high permeability of blood vessels is that exposure to blood plasma increases bone marrow HSPC ROS levels, augmenting their migration and differentiation, while compromising their long-term repopulation and survival. These findings may have relevance for clinical haematopoietic stem cell transplantation and mobilization protocols.

Antiangiogenesis-based cancer therapies, specifically those targeting the VEGF-A/VEGFR2 pathway, have been approved for subsets of solid tumors. However, these therapies result in an increase in hematologic adverse events. We surmised that both the bone marrow vasculature and VEGF receptor-positive hematopoietic cells could be impacted by VEGF pathway-targeted therapies. We used a mouse model of spontaneous breast cancer to decipher the mechanism by which VEGF pathway inhibition alters hematopoiesis. Tumor-bearing animals, while exhibiting increased angiogenesis at the primary tumor site, showed signs of shrinkage in the sinusoidal bone marrow vasculature accompanied by an increase in the hematopoietic stem cell-containing Lin-cKit(+)Sca1(+) (LKS) progenitor population. Therapeutic intervention by targeting VEGF-A, VEGFR2, and VEGFR3 inhibited tumor growth, consistent with observed alterations in the primary tumor vascular bed. These treatments also displayed systemic effects, including reversal of the tumor-induced shrinkage of sinusoidal vessels and altered population balance of hematopoietic stem cells in the bone marrow, manifested by the restoration of sinusoidal vessel morphology and hematopoietic homeostasis. These data indicate that tumor cells exert an aberrant systemic effect on the bone marrow microenvironment and VEGF-A/VEGFR targeting restores bone marrow function., (©2015 American Association for Cancer Research.)

Understanding the intricate cellular components of the bone marrow microenvironment can lead to the discovery of novel extrinsic factors that are responsible for the initiation and progression of leukemic disease. We have shown that endothelial cells (ECs) provide a fertile niche that allows for the propagation of primitive and aggressive leukemic clones. Activation of the ECs by vascular endothelial growth factor (VEGF)-A provides cues that enable leukemic cells to proliferate at higher rates and also increases the adhesion of leukemia to ECs. Vascular endothelial growth factor A-activated ECs decrease the efficacy of chemotherapeutic agents to target leukemic cells. Inhibiting VEGF-dependent activation of ECs by blocking their signaling through VEGF receptor 2 increases the susceptibility of leukemic cells to chemotherapy. Therefore, the development of drugs that target the activation state of the vascular niche could prove to be an effective adjuvant therapy in combination with chemotherapeutic agents., (Copyright © 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Myelosuppression is a life-threatening complication of antineoplastic therapy, but treatment is restricted to a few cytokines with unilineage hematopoietic activity. Although hematopoietic stem cells (HSCs) are predominantly quiescent during homeostasis, they are rapidly recruited into cell cycle by stresses, including myelosuppressive chemotherapy. Factors that induce HSCs to proliferate during stress have been characterized, but it is not known how HSC quiescence is then reestablished. In this study, we show that TGFβ signaling is transiently activated in hematopoietic stem and progenitor cells (HSPCs) during hematopoietic regeneration. Blockade of TGFβ signaling after chemotherapy accelerates hematopoietic reconstitution and delays the return of cycling HSCs to quiescence. In contrast, TGFβ blockade during homeostasis fails to induce cycling of HSPCs. We identified the cyclin-dependent kinase inhibitor Cdkn1c (p57) as a key downstream mediator of TGFβ during regeneration because the recovery of chimeric mice, incapable of expressing p57 in HSPCs, phenocopies blockade of TGFβ signaling after chemotherapy. This study demonstrates that context-dependent activation of TGFβ signaling is central to an unrecognized counterregulatory mechanism that promotes homeostasis once hematopoiesis has sufficiently recovered from myelosuppressive chemotherapy. These results open the door to new, potentially superior, approaches to promote multilineage hematopoietic recovery by blocking the TGFβ signaling that dampens regeneration.

Background: Circulating Endothelial Progenitor Cell (EPC) levels are reduced in diabetes mellitus. This may be a consequence of impaired mobilization of EPC from the bone marrow. We hypothesized that under diabetic conditions, mobilization of EPC from the bone marrow to the circulation is impaired -at least partly- due to dysfunction of the bone marrow stromal compartment., Methods: Diabetes was induced in mice by streptozotocin injection. Circulating Sca-1(+)Flk-1(+) EPC were characterized and quantified by flow cytometry at baseline and after mobilization with G-CSF/SCF injections. In vivo hemangiogenic recovery was tested by 5-FU challenge. Interaction within the bone marrow environment between CD34(+) hematopoietic progenitor cells (HPC) and supporting stroma was assessed by co-cultures. To study progenitor cell-endothelial cell interaction under normoglycemic and hyperglycemic conditions, a co-culture model using E4Orf1-transfected human endothelial cells was employed., Results: In diabetic mice, bone marrow EPC levels were unaffected. However, circulating EPC levels in blood were lower at baseline and mobilization was attenuated. Diabetic mice failed to recover and repopulate from 5-FU injection. In vitro, primary cultured bone marrow stroma from diabetic mice was impaired in its capacity to support human CFU-forming HPC. Finally, hyperglycemia hampered the HPC supportive function of endothelial cells in vitro., Conclusion: EPC mobilization is impaired under experimental diabetic conditions and our data suggest that diabetes induces alterations in the progenitor cell supportive capacity of the bone marrow stroma, which could be partially responsible for the attenuated EPC mobilization and reduced EPC levels observed in diabetic patients.

Sinusoidal endothelial cells (SECs) comprise the platform where trafficking into and out of the BM occurs and where hematopoietic stem and progenitor cells (HSPC) harbor and receive cues for self-renewal, survival, and differentiation. Therefore, SECs are referred to as a bone marrow vascular niche (BMVN). Hematopoietic regeneration has been shown to occur only with concurrent angiogenic regeneration. However, there are still not sufficient means to identify and isolate SECs, therefore the "niche endothelial cell" remains incompletely characterized. VEGF-receptor-3 (VEGFR3) is expressed exclusively by the SECs, while Sca1 and Tie2 are only expressed on the VEGFR3(-) arteriolar endothelium. We previously demonstrated the importance of vascular recovery in hematopoietic regeneration from myelosuppression due to cytotoxic agents or whole-body irradiation. Therefore to establish the functional importance of SECs, the mechanisms underlying BMVN regeneration were examined utilizing a 5-fluorouracil (5-FU) myelosuppression model of vascular damage. Injection of antibodies against murine VEGFR-1 and -2 had no significant effect on hemangiogenic recovery. However, when soluble VEGFR-1, a decoy receptor for VEGF-A and PlGF, was injected after 5-FU, both angiogenic remodeling and regeneration of megakaryopoiesis were delayed. In conclusion, we show that the bone marrow vasculature comprises heterogeneous compartments. SECs are distinguished from arterioles by unique immunophenotypes. Regeneration of damaged SECs is the rate-limiting step in hematopoietic regeneration from myelosuppressive therapy. Novel, high-efficiency VEGF-binding drugs in combination with chemotherapeutic agents may lead to cases of prolonged cytopenia.

Myelosuppression damages the bone marrow (BM) vascular niche, but it is unclear how regeneration of bone marrow vessels contributes to engraftment of transplanted hematopoietic stem and progenitor cells (HSPCs) and restoration of hematopoiesis. We found that chemotherapy and sublethal irradiation induced minor regression of BM sinusoidal endothelial cells (SECs), while lethal irradiation induced severe regression of SECs and required BM transplantation (BMT) for regeneration. Within the BM, VEGFR2 expression specifically demarcated a continuous network of arterioles and SECs, with arterioles uniquely expressing Sca1 and SECs uniquely expressing VEGFR3. Conditional deletion of VEGFR2 in adult mice blocked regeneration of SECs in sublethally irradiated animals and prevented hematopoietic reconstitution. Similarly, inhibition of VEGFR2 signaling in lethally irradiated wild-type mice rescued with BMT severely impaired SEC reconstruction and prevented engraftment and reconstitution of HSPCs. Therefore, regeneration of SECs via VEGFR2 signaling is essential for engraftment of HSPCs and restoration of hematopoiesis.

Thrombopoietic cells may differentially promote or inhibit tissue vascularization by releasing both pro- and antiangiogenic factors. However, the molecular determinants controlling the angiogenic phenotype of thrombopoietic cells remain unknown. Here, we show that expression and release of thrombospondins (TSPs) by megakaryocytes and platelets function as a major antiangiogenic switch. TSPs inhibited thrombopoiesis, diminished bone marrow microvascular reconstruction following myelosuppression, and limited the extent of revascularization in a model of hind limb ischemia. We demonstrate that thrombopoietic recovery following myelosuppression was significantly enhanced in mice deficient in both TSP1 and TSP2 (TSP-DKO mice) in comparison with WT mice. Megakaryocyte and platelet levels in TSP-DKO mice were rapidly restored, thereby accelerating revascularization of myelosuppressed bone marrow and ischemic hind limbs. In addition, thrombopoietic cells derived from TSP-DKO mice were more effective in supporting neoangiogenesis in Matrigel plugs. The proangiogenic activity of TSP-DKO thrombopoietic cells was mediated through activation of MMP-9 and enhanced release of stromal cell-derived factor 1. Thus, TSP-deficient thrombopoietic cells function as proangiogenic agents, accelerating hemangiogenesis within the marrow and revascularization of ischemic hind limbs. As such, interference with the release of cellular stores of TSPs may be clinically effective in augmenting neoangiogenesis.

The bone marrow vascular niche consists of a network of thin-walled and fenestrated sinusoidal vessels whose integrity is maintained and supported by surrounding hematopoietic cells. However, this dependence is highly reciprocal in that the bone marrow vasculature provides not only a conduit for mature hematopoietic cells to the peripheral circulation but also a site where hematopoietic progenitors, especially megakaryocytes, differentiate and set the stage for full reconstitution of hematopoiesis.

Stem cells reside in a physical niche, a particular microenvironment. The organization of cellular niches has been shown to play a key role in regulating normal stem cell differentiation, maintenance and regeneration. Hematopoietic stem cells (HSC) emerge at distinct allocation territories during ontogenesis, notably the aorto-gonadal region, the fetal liver. Adult HSC expand and differentiate exclusively in the bone marrow (BM). They can be mobilized into the blood stream. This implies that stem cells are not autonomous units of development; rather, tissue specific niches control their destiny. Interaction of HSCs with their stem cell niches is critical for adult hematopoiesis in the BM. A niche is composed of stromal cells, which either through direct cell-to-cell contact or via release of soluble factors maintain the typical features of stem cells, mainly stem cell quiescence, maintenance or expansion. HSCs are keeping the balance of the quiescence and the self-renewal in the stem cell niche, and are maintaining long-term hematopoiesis.Therefore, an understanding of cellular and chemical architecture of the stem cell niche is vital in understanding stem cell behavior. This review summarizes the recent developments in our understanding of the stem cell niche with particular focus on the HSC niche.

The molecular pathways involved in the differentiation of hematopoietic progenitors are unknown. Here we report that chemokine-mediated interactions of megakaryocyte progenitors with sinusoidal bone marrow endothelial cells (BMECs) promote thrombopoietin (TPO)-independent platelet production. Megakaryocyte-active cytokines, including interleukin-6 (IL-6) and IL-11, did not induce platelet production in thrombocytopenic, TPO-deficient (Thpo(-/-)) or TPO receptor-deficient (Mpl(-/-)) mice. In contrast, megakaryocyte-active chemokines, including stromal-derived factor-1 (SDF-1) and fibroblast growth factor-4 (FGF-4), restored thrombopoiesis in Thpo(-/-) and Mpl(-/-) mice. FGF-4 and SDF-1 enhanced vascular cell adhesion molecule-1 (VCAM-1)- and very late antigen-4 (VLA-4)-mediated localization of CXCR4(+) megakaryocyte progenitors to the vascular niche, promoting survival, maturation and platelet release. Disruption of the vascular niche or interference with megakaryocyte motility inhibited thrombopoiesis under physiological conditions and after myelosuppression. SDF-1 and FGF-4 diminished thrombocytopenia after myelosuppression. These data suggest that TPO supports progenitor cell expansion, whereas chemokine-mediated interaction of progenitors with the bone marrow vascular niche allows the progenitors to relocate to a microenvironment that is permissive and instructive for megakaryocyte maturation and thrombopoiesis. Progenitor-active chemokines offer a new strategy to restore hematopoiesis in a clinical setting.

Angiogenic defects in Id mutant mice inhibit the growth of tumor xenografts, providing a genetic model for antiangiogenic stress. Our work tests the consequences of such stress on progression of more physiological Pten+/- tumors. While tumor growth occurs despite impaired angiogenesis, disruption of vasculature by Id loss causes tumor cells to experience hypoxia and necrosis, the extent of which is tumor dependent. We show that bone-marrow-derived endothelial precursors contribute functionally to neovasculature of some but not all Pten+/- tumors, partially rescuing Id mutant phenotype. We demonstrate that loss of Id1 in tumor endothelial cells results in downregulation of several proangiogenic genes, including alpha6 and beta4 integrins, matrix metalloprotease-2, and fibroblast growth factor receptor-1. Inhibition of these factors phenocopies loss of Id in in vivo angiogenesis assays.

The mechanism by which angiogenic factors recruit bone marrow (BM)-derived quiescent endothelial and hematopoietic stem cells (HSCs) is not known. Here, we report that functional vascular endothelial growth factor receptor-1 (VEGFR1, Flt-1) is expressed on a subpopulation of human CD34(+) and mouse Lin-Sca-1(+)c-Kit(+) BM-repopulating stem cells, conveying signals for recruitment of HSCs and reconstitution of hematopoiesis. Inhibition of VEGFR1 signaling, but not VEGFR2 (Flk-1, KDR), blocked HSC cell cycling, differentiation and hematopoietic recovery after BM suppression, resulting in the demise of the treated mice. Plasma elevation of placental growth factor (PlGF), which signals through VEGFR1, but not VEGFR2, restored hematopoiesis during the early and late phases following BM suppression. The mechanism whereby PlGF enhanced early phases of BM recovery was mediated directly through rapid chemotaxis of readily available VEGFR1(+) BM-repopulating and progenitor cells. The late phase of hematopoietic recovery was driven by PlGF-induced upregulation of matrix metalloproteinase-9 (MMP-9) in the BM, mediating the release of soluble Kit-ligand (sKitL). sKitL increased proliferation and motility of HSCs and progenitor cells, thereby augmenting hematopoietic recovery. PlGF promotes recruitment of VEGFR1(+) HSCs from a quiescent to a proliferative microenvironment within the BM, favoring differentiation, mobilization, and reconstitution of hematopoiesis.

Stem cells within the bone marrow (BM) exist in a quiescent state or are instructed to differentiate and mobilize to circulation following specific signals. Matrix metalloproteinase-9 (MMP-9), induced in BM cells, releases soluble Kit-ligand (sKitL), permitting the transfer of endothelial and hematopoietic stem cells (HSCs) from the quiescent to proliferative niche. BM ablation induces SDF-1, which upregulates MMP-9 expression, and causes shedding of sKitL and recruitment of c-Kit+ stem/progenitors. In MMP-9-/- mice, release of sKitL and HSC motility are impaired, resulting in failure of hematopoietic recovery and increased mortality, while exogenous sKitL restores hematopoiesis and survival after BM ablation. Release of sKitL by MMP-9 enables BM repopulating cells to translocate to a permissive vascular niche favoring differentiation and reconstitution of the stem/progenitor cell pool.

Although thrombopoietin has been shown to promote megakaryocyte (MK) proliferation and maturation, the exact mechanism and site of platelet formation are not well defined. Studies have shown that MKs may transmigrate through bone marrow endothelial cells (BMEC), and release platelets within the sinusoidal space or lung capillaries. In search for chemotactic factor(s) that may mediate transmigration of MKs, we have discovered that mature polyploid MKs express the G protein-coupled chemokine receptor CXCR4 (Fusin, LESTR). Therefore, we explored the possibility that stromal cell-derived factor 1 (SDF-1), the ligand for CXCR4, may also induce transendothelial migration of mature MKs. SDF-1, but not other CXC or CC chemokines, was able to mediate MK migration (ED50 = 125 pmol/liter). The MK chemotaxis induced by SDF-1 was inhibited by the CXCR4-specific mAb (12G5) and by pertussis toxin, demonstrating that signaling via the G protein-coupled receptor CXCR4 was necessary for migration. SDF-1 also induced MKs to migrate through confluent monolayers of BMEC by increasing the affinity of MKs for BMEC. Activation of BMEC with interleukin 1beta resulted in a threefold increase in the migration of MKs in response to SDF-1. Neutralizing mAb to the endothelial-specific adhesion molecule E-selectin blocked the migration of MKs by 50%, suggesting that cellular interaction of MKs with BMEC is critical for the migration of MKs. Light microscopy and ploidy determination of transmigrated MKs demonstrated predominance of polyploid MKs. Virtually all platelets generated in the lower chamber also expressed CXCR4. Platelets formed in the lower chamber were functional and expressed P-selectin (CD62P) in response to thrombin stimulation. Electron microscopy of the cells that transmigrated through the BMEC monolayers in response to SDF-1 demonstrated the presence of intact polyploid MKs as well as MKs in the process of platelet formation. These results suggest that SDF-1 is a potent chemotactic factor for mature MKs. Expression of CXCR4 may be the critical cellular signal for transmigration of MKs and platelet formation.

We have shown that bone marrow microvascular endothelial cells (BMEC) support growth and differentiation of haemopoietic progenitors in vitro by elaboration of haemopoietic cytokines. Since generation of eosinophils can be observed in these coculture experiments, and BMEC do not produce interleukin (IL)-3, we evaluated BMEC for expression of IL-5, a specific growth factor for the eosinophilic lineage. Using RT-PCR, IL-5 mRNA was expressed by BMEC after stimulation (12 h) with lipopolysaccharide (LPS), IL-1, IL-2 and phorbol myristate acetate (PMA), but not by resting BMEC, after stimulation with TNF-alpha or interferon (IFN)-gamma. Moreover, IFN-gamma suppressed expression of IL-5 in response to LPS and IL-2. The identity of the PCR products was confirmed by restriction enzyme digestion, which resulted in fragments of the predicted size. T lymphocytes were not present in the endothelial cultures as demonstrated by absence of CD2 mRNA. Using a sensitive (1 pg/ml) ELISA assay. IL-5 was detected after 48 h incubation of BMEC with IL-2 (4.1 pg/10(6) cells) or with a combination of LPS and IL-2 (4.8 pg). However, the number of eosinophils generated after 4 weeks coculture of CD34+ haemopoietic cells with BMEC was not increased by addition of IL-2. RT-PCR revealed that BMEC in coculture with haemopoietic cells expressed IL-5 even without addition of exogenous cytokines or stimulating agents. In conclusion, expression of IL-5 by BMEC can be stimulated by cytokines (IL-1, IL-2), LPS, PMA, and coculture with proliferating haemopoietic cells. Thus, BMEC may support proliferation and differentiation of eosinophils in the bone marrow. IFN-gamma represents a cytokine with an inhibitory effect on IL-5 expression by BMEC. In addition, eosinophilia in response to circulating IL-2 or bacterial products (LPS) in vivo may be partially mediated by BMEC or vascular endothelium.

Bone marrow microvascular endothelial cells (BMEC) are a functional component of the bone marrow stroma and have been shown to release hematopoietic regulatory factors as well as to selectively adhere and support the proliferation and differentiation of CD34+ hematopoietic progenitors. An early passage of these cells was immortalized by transfection with a vector (pSVT) encoding the large T antigen of SV40. The transformed cell line (CDC/CU.BMEC-1) expresses the SV40 transcript, retains the primary cell expression of Ulex europeaus and vWF/ FVIII, and incorporates acetylated low-density lipoprotein. In addition, BMEC-1 mirrors the phenotype of the primary cells with only a few exceptions. Both cell populations express the cellular adhesion molecules ICAM-1 and PECAM and also VCAM-1 and ELAM-1 after upregulation by tumor necrosis factor-alpha. The fibronectin receptor, hyaluronate receptor, collagen receptor, integrins VLA-alpha 3, VLA-alpha 4, and beta 4, endoglin, collagen IV, CD58, and CD61 are also expressed. The only differences are that BMEC-1 expresses higher levels of ICAM-1, CD58, CD34, CD36, and c-kit than the primary cells. The supernatants of primary cell and BMEC-1 contain stem cell factor, interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1 alpha, IL-11, and G-CSF. The functional significance of these hematopoietic cytokines was demonstrated in transwell cultures. Both cell populations supported the expansion of progeny from CD34+ cell-enriched cord blood mononuclear cells suspended in the upper chamber. These characteristics, plus the fact that BMEC-1 can be maintained independently of exogenous growth factors and exhibit contact inhibition, indicate that this cell line can be used to further define the role of BMEC in hematopoiesis.

Endothelial cells are a major component of the bone marrow (BM) microenvironment that regulate the trafficking and homing of hematopoietic progenitor and stem cells. In this paper, we provide evidence that BM endothelial cells (BMECs) also support multilineage hematopoiesis by elaboration of soluble cytokines. Hematopoietic progenitor cells incubated in direct contact with BMEC monolayers, or physically separated by microporous membrane, expanded five-fold to sevenfold at 7 days, in the absence of exogenous cytokines. Flow cytometric analysis of proliferating progenitor cells grown in the presence of BMEC monolayers showed that by day 14 of coculture, 70% to 80% of hematopoietic cells were myeloid, expressing CD15 or CD14, and 14% to 19% were megakaryocytic, expressing GPIIb/IIIa or GPIb. CD34+ cells derived from umbilical cord blood, cultured in the upper chamber of transwell culture plates, as well as the cells grown in direct contact with BMEC monolayers, generated progenitors for up to 70 days. Unstimulated BMEC monolayers constitutively produce interleukin-6, Kit-ligand, granulocyte colony-stimulating factor, and granulocyte macrophage colony-stimulating factor. These data suggest that BMEC regulate proliferation of hematopoietic progenitor cells and long-term culture initiating cells by elaboration of lineage-specific cytokines.