The mechanisms of hematopoietic progenitor cell egress and clinical mobilization are not fully understood. Herein, we report that in vivo desensitization of Sphingosine-1-phosphate (S1P) receptors by FTY720 as well as disruption of S1P gradient toward the blood, reduced steady state egress of immature progenitors and primitive Sca-1 ؉ /c-Kit ؉ /Lin ؊ (SKL) cells via inhibition of SDF-1 release. Administration of AMD3100 or G-CSF to mice with deficiencies in either S1P production or its receptor S1P 1 , or pretreated with FTY720, also resulted in reduced stem and progenitor cell mobilization. Mice injected with AMD3100 or G-CSF demonstrated transient increased S1P levels in the blood mediated via mTOR signaling, as well as an elevated rate of immature c-Kit ؉ /Lin ؊ cells expressing surface S1P 1 in the bone marrow (BM). Importantly, we found that S1P induced SDF-1 secretion from BM stromal cells including Nestin ؉ mesenchymal stem cells via reactive oxygen species (ROS) signaling. Moreover, elevated ROS production by hematopoietic progenitor cells is also regulated by S1P. Our findings reveal that the S1P/S1P 1 axis regulates progenitor cell egress and mobilization via activation of ROS signaling on both hematopoietic progenitors and BM stromal cells, and SDF-1 release. The dynamic cross-talk between S1P and SDF-1 integrates BM stromal cells and hematopoeitic progenitor cell motility. (Blood. 2012;119(11):2478-2488) IntroductionMotility is a key feature of hematopoietic stem and progenitor cells (HSPCs). These cells are continuously released at basal levels from the bone marrow (BM) reservoir to the circulation during steady state homeostasis together with maturing leukocytes, and at increased rates on stress situations, such as bleeding or inflammation. 1,2 The complex process of HSPC trafficking is orchestrated by various cytokines, chemokines, proteolytic enzymes, and adhesion molecules 3-5 through a dynamic interplay between the immune and nervous systems within the bone microenvironment. 1,2,6-8 HSPC mobilization can be clinically induced by a variety of cytokines and drugs, such as granulocyte colony stimulating factor (G-CSF, the most commonly used agent), 9,10 sulfated polysaccharides, 11,12 and recently also by AMD3100. 13,14 Repetitive G-CSF administrations cause mobilization by inducing proliferation and differentiation of HSPC, thus increasing their pool size, accompanied by reduced retention in the BM microenvironment. 15 The chemokine stromal cell-derived factor-1 (SDF-1, also termed CXCL12), which is the most powerful chemoattractant of both human and murine HSPCs, 16,17 and its major receptor CXCR4 are key players in HSPC mobilization. 10,12,13,[18][19][20][21] SDF-1 is transiently increased in the murine BM during G-CSF stimulation followed by its downregulation at both protein 18,22 and mRNA 23 levels, reaching a nadir at the peak of HSPC mobilization. 18 The intensified SDF-1/CXCR4 interactions induce enhanced production of reactive oxygen species (ROS) through activation of the HGF/c-Met path...
Ceramide-1-phosphate (C1P) is a bioactive lipid that, in contrast to ceramide, is an anti-apoptotic molecule released from cells that are damaged and “leaky”. As reported recently, C1P promotes migration of hematopoietic cells. In the current paper, we tested the hypothesis that C1P released upon tissue damage may play an underappreciated role in chemoattraction of various types of stem cells and endothelial cells involved in tissue/organ regeneration. We show for a first time that C1P is upregulated in damaged tissues and chemoattracts BM-derived multipotent stroma cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic-like stem cells (VSELs). Furthermore, compared to other bioactive lipids, C1P more potently chemoattracted human umbilical vein endothelial cells (HUVECs) and stimulated tube formation by these cells. C1P also promoted in vivo vascularization of Matrigel implants and stimulated secretion of stromal derived factor-1 (SDF-1) from BM-derived fibroblasts. Thus, our data demonstrate, for the first time, that C1P is a potent bioactive lipid released from damaged cells that potentially plays an important and novel role in recruitment of stem/progenitor cells to damaged organs and may promote their vascularization.
Hunter syndrome is an X-linked lysosomal storage disease caused by a deficiency in the enzyme iduronate-2-sulfatase (IDS), leading to the accumulation of glycosaminoglycans (GAGs). Two recombinant enzymes, idursulfase and idursulfase beta are currently available for enzyme replacement therapy for Hunter syndrome. These two enzymes exhibited some differences in various clinical parameters in a recent clinical trial. Regarding the similarities and differences of these enzymes, previous research has characterized their biochemical and physicochemical properties. We compared the in vitro and in vivo efficacy of the two enzymes on patient fibroblasts and mouse model. Two enzymes were taken up into the cell and degraded GAGs accumulated in fibroblasts. In vivo studies of two enzymes revealed similar organ distribution and decreased urinary GAGs excretion. Especially, idursulfase beta exhibited enhanced in vitro efficacy for the lower concentration of treatment, in vivo efficacy in the degradation of tissue GAGs and improvement of bones, and revealed lower anti-drug antibody formation. A biochemical analysis showed that both enzymes show largely a similar glycosylation pattern, but the several peaks were different and quantity of aggregates of idursulfase beta was lower.
BackgroundMucopolysaccharidosis type I (MPS I) is caused by the deficiency of alpha-L-iduronidase (IDUA), which is involved in the degradation of glycosaminoglycans (GAGs), such as heparan sulfate and dermatan sulfate in the lysosome. It has been reported that joint symptoms are almost universal in MPS I patients, and even in the case of attenuated disease, they are the first symptom that brings a child to medical attention. However, functional tests and biological markers have not been published for the evaluation of the limitations in joint and locomotion in animal model-mimicking MPS.MethodsWe generated IDUA knockout (KO) mice to observe whether they present impairment of joint function. KO mice were characterized phenotypically and tested dual-energy X-ray absorptiometry analysis (DEXA), open-field, rotarod, and grip strength.ResultsThe IDUA KO mice, generated by disruption between exon 6 and exon 9, exhibited clinical and laboratory findings, such as high urinary GAGs excretion, GAGs accumulation in various tissues, and significantly increased bone mineral density (BMD) in both female and male mice in the DEXA of the femur and whole bone. Remarkably, we observed a decrease in grasp function, decreased performance in the rotarod test, and hypo-activity in the open-field test, which mimic the limitations of joint mobility and decreased motor performance in the 6-min walk test in patients with MPS I.ConclusionsWe generated a new IDUA KO mouse, tested open field, rotarod and grip strength and demonstrated decrease in grip strength, decreased performance and hypo-activity, which may be useful for investigating therapeutic approaches, and studying the pathogenesis of joint and locomotion symptoms in MPS I.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-015-0337-3) contains supplementary material, which is available to authorized users.
1247 Background. Hematopoietic stem progenitor cells (HSPCs) are retained in bone marrow (BM) niches due to the stromal-derived growth factor-1 (SDF-1)–CXCR4 receptor axis and interactions between Very Late Antigen-4 (VLA-4, also known as α 4β1integrin) and its ligand, Vascular Adhesion Molecule-1 (VCAM-1 or CD106). While HSPCs express CXCR4 and VLA-4, their corresponding ligands, SDF-1 and VCAM-1, are expressed by cells in the BM microenvironment (e.g., osteoblasts and fibroblasts). While a role for the SDF-1–CXCR4 axis in the retention of HSPCs in BM under steady-state conditions is undisputed, its role in stem cell homing needs further clarification. For many years, it has been assumed that the chemotactic SDF-1 gradient across the BM–peripheral blood (PB) barrier determines whether cells home from PB into the BM microenvironment. However, this simple explanation has been challenged by several observations supporting the existence of SDF-1–CXCR4-independent homing mechanisms. For example, i) CXCR4−/− fetal liver HSPCs can home to BM in an SDF-1-independent manner, ii) homing of murine HSPCs made refractory to SDF-1 by preincubation and co-injection with a CXCR4 receptor antagonist (AMD3100) is normal or only mildly reduced, iii) HSPCs in which CXCR4 expression is reduced by a SDF-1 intrakine strategy remain able to engraft in lethally irradiated recipients, and, as we recently reported, iv) myeloablative conditioning for transplantation induces a highly proteolytic microenvironment in BM that leads to proteolytic degradation of SDF-1 (Cancer Res. 2010;70:3402, Leukemia. 2012;26:106). Aim of the study. Based on these observations strongly suggesting the involvement of other factors and/or supportive mechanisms in the SDF-1-mediated homing of HSPCs, we became interested in identifying these unknown factors, which support homing of HSPCs when SDF-1 is degraded in the proteolytic microenvironment of BM or even if it is completely absent. Experimental approach. We tested several growth factors, cytokines, bioactive lipids, extracellular nucleotides, and antimicrobial cationic peptides for their potential involvement in homing by employing i) Transwell migration assays and ii) signaling studies in the presence or absence of specific inhibitors. We studied the chemotactic responsiveness of these factors against BM, umbilical cord blood (UCB), and mobilized peripheral blood (mPB) cells. We also focused on the molecular mechanisms responsible for the observed phenomena. Salient observations. Out of >50 different factors tested in addition to SDF-1, only sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), ATP, UTP, and GTP, which are released by cells after BM microenvironment damaged by radio/chemotherapy conditioning for transplantation, were able to chemoattract BM-purified HSPCs. The responsiveness of HSPCs to these factors was inhibited after exposure to pertussis toxin (PTX) and after inhibiting MAPKp42/44 and AKT. Interestingly, in contrast to BM-isolated HSPCs, the chemotactic responsiveness of UCB and mPB HSPCs to these factors was significantly weaker, which suggests desensitization of the corresponding receptors by factors already present in peripheral blood plasma. We also found that BM stroma exposed to myeloablative doses of radio-chemotherapy secretes two antimicrobial cationic peptides, LL-37 and β2-defensin, that, while not direct chemottractants for HSPCs, strongly enhance the responsiveness of HSPCs to an SDF-1 gradient. This phenomenon plays a crucial role in situations in which the SDF-1 homing gradient is impaired by a highly proteolytic BM microenvironment after myeloablative conditioning for transplantation. Conclusions. Since all these direct chemottractants and priming factors are upregulated in BM after myeloblative conditioning for transplantation, a more complex picture of homing emerges that involves several factors that support, and in some situations even replace, the SDF-1–CXCR4 axis. We also conclude that the priming effects by LL-37 and β2-defensin play a critical role in homing of UCB- and mPB-derived HSPCs, which respond robustly to an SDF-1 gradient. Moreover, data in an animal model lend further support to this concept. Disclosures: No relevant conflicts of interest to declare.
2957 Background. Hematopoietic stem progenitor cells (HSPCs) are retained in bone marrow (BM) niches due to the stromal-derived growth factor-1 (SDF-1)–CXCR4 receptor axis and interactions between Very Late Antigen-4 (VLA-4 or a4b1 integrin) and its ligand, Vascular Adhesion Molecule-1 (VCAM-1 or CD106). While HSPCs express CXCR4 and VLA-4, their corresponding ligands, SDF-1 and VCAM-1, are expressed by cells in the BM microenvironment (e.g., osteoblasts and fibroblasts). While a role for the SDF-1–CXCR4 axis in the retention of HSPCs in BM under steady state conditions is undisputed, its role in stem cell homing needs further clarification. For many years, it has been proposed that concentration differences between the chemotactic SDF-1 gradient across the BM–peripheral blood (PB) barrier determines whether cells will home from PB into the BM microenvironment. However, this simple explanation has been challenged by several observations supporting the existence of SDF-1–CXCR4-independent homing mechanisms. For example, i) CXCR4−/– fetal liver HSPCs can home to BM in an SDF-1-independent manner, ii) homing of murine HSPCs made refractory to SDF-1 by preincubation and co-injection with a CXCR4 receptor antagonist (AMD3100) is normal or only mildly reduced, iii) HSPCs in which CXCR4 expression is reduced by a SDF-1 intrakine strategy remain able to engraft in lethally irradiated recipients, and as we recently reported, iv) myeloablative conditioning for transplantation induces a highly proteolytic microenvironment in BM that leads to proteolytic degradation of SDF-1 (Leukemia 2011, doi: 10.1038/leu.2011.185). Aim of the study. Based on these observations that strongly suggest the involvement of other factors and/or supportive mechanisms in the SDF-1-mediated homing of HSPCs we become interested in a role of bioactive lipids (sphingosine-1 phosphate; S1P and ceramide-1 phosphate; C1P) as well as priming phenomenon of SDF-1-CXCR4 axis by cationic antimicrobial peptides (CAMPs) in HSPCs homing. Results. We present evidence that gradients of bioactive lipids, such as S1P and C1P, which are products of membrane lipid metabolism, are involved in stem cell lodgment into BM. In addition, we present another important mechanism that enhances stem cell response to an low SDF-1 concentration gradient, namely, the “priming effect”. This mechanism is based on a phenomenon in which CAMPs such as complement cascade cleavage fragment C3a and stroma fibroblast- and granulocytes-derived antimicrobial peptide LL-37 significantly increase (up to 25 times) the chemotactic responsiveness of HSPCs to very low “tissue concentration-relevant” SDF-1 gradients (1–3 ng/ml). To support this further murine HSPCs primed ex vivo with CAMPs engrafted by 3–5 days faster in lethally irradiated littermates. At the molecular level, this sensitization of the responsiveness to SDF-1 after exposure to CAMPs depends on the incorporation of the CXCR4 receptor into membrane lipid rafts. This phenomenon is specific for SDF-CXCR4 axis as we did not observe that this phenomenon influences S1P- and C1P-mediated in vitro chemotaxis. Overall, these changes of SDF-1, S1P, and C1P chemotactic gradients and the appearance of priming factors in the BM microenvironment and PB plasma are triggered during conditioning for transplantation by radio-chemotherapy by the induction of a proteolytic microenvironment in BM and the activation of the complement cascade (CC). Conclusions. We provide in vivo evidence that C3a and LL-37, which have primarily antimicrobial functions and are harmless to mammalian cells, could be clinically applied to accelerate engraftment as ex vivo priming agent for transplanted human HSPCs. This novel approach based on a short ex vivo incubation of HSPCs in the graft before transplantation by CAMPs (C3a and LL-37) is of particular important in umbilical cord blood transplantations, where the number of available HSPCs in single unit for transplant is limited. This strategy is currently tested in the clinic. Disclosures: No relevant conflicts of interest to declare.
554 The stromal derived factor-1 (SDF-1)–CXCR4 axis plays an unquestioned role in developmental migration of hematopoietic stem cells (HSPCs) and their retention in the bone marrow (BM). However, changes in the SDF-1 gradient between BM and peripheral blood (PB) do not always support its having a crucial role as chemoattractant for mobilization or homing of HSPCs. As demonstrated by others (e.g., Bone Marrow Transplantation 2003; 31:651–654, and Transfus Apher Sci 2009;40:159) and us (Leukemia 2010;24:976–985) the plasma SDF-1 level does not correlate with mobilization of HSPCs. On the other hand, there is increasing doubt about an exclusive role for SDF-1 in homing of HSPCs in BM. This is based on evidence that i) CXCR4−/− fetal liver HSPCs may home to BM in an SDF-1–independent manner (Immunity 1999;10:463-471), ii) homing of murine HSPCs made refractory to SDF-1 by incubation and co-injection with a CXCR4 receptor antagonist is normal or only mildly reduced (Science 2004;305:1000), and finally iii) HSPCs in which CXCR4 has been knocked down by means of an SDF-1 intrakine strategy also engraft in lethally irradiated recipients (Blood 2000;96:2074–,2080). All this strongly suggests the existence of other factors involved in the mobilization and homing of HSPCs. Moreover, while SDF-1 is a potent chemoattractant for HSPCs when employed at supraphysiological concentrations in vitro, as a peptide it is highly susceptible to degradation by proteases that are elevated, for example, in PB during stem cell mobilization or in the BM microenvironment after myeloablative conditioning for transplantation. Employing ELISA for detection in the present study, we observed insignificant changes in SDF-1 level both in PB during mobilization and in BM after myeloablative conditioning. We also found that mobilized PB (mPB) plasma as well as conditioned media (CM) from lethally irradiated mice chemoattract HSPCs in an SDF-1–independent manner as demonstrated by i) normal chemotaxis of AMD3100 pre-treated cells and ii) preservation of chemotactic activity of plasma and BM-derived CM following heat inactivation. However, the chemotactic activity of mPB plasma and BM CM was inhibited after stripping by activated charcoal. This suggested the involvement of small molecule bioactive lipids. It is known that sphingolipids, which are important components of cell membranes, give rise to two bioactive derivatives, sphingosine-1 phosphate (S1P) and ceramide-1 phosphate (C1P), with S1P already identified as a chemoattractant for HSPCs (Ann N Y Acad Sci. 200;1044:84–89). To our surprise, we found that C1P is also a strong chemoattractant for human and murine HSPCs. In addition, we observed that at physiological concentrations both these bioactive lipids i) activate phosphorylation of MAPKp42/44 and AKT in HSPCs, ii) induce expression of matrix metalloproteinases (MMPs), and iii) modulate adhesion to stroma and endothelium. Interestingly, by employing ELISA and/or mass spectophotometry we found that, while the S1P level increases in PB during mobilization, the C1P level increases in BM after myeloablative conditioning for transplantation. Based on these findings, we propose a new paradigm in which the S1P:C1P ratio plays a role in mobilization and homing of HSPCs. While S1P is a major chemoattractant that directs egress of HSPCs from BM into PB, C1P released from damaged cells in BM after myeloablative conditioning creates a homing gradient for circulating HSPCs. We also postulate that the S1P:C1P ratio plays a more universal role and is involved in regulating migration of other types of stem cells, such as circulating mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic-like (VSEL) stem cells. Accordingly, while S1P plays a role in egress of stem cells into PB, C1P released from damaged cells (e.g., in infarcted myocardium or brain tissue after stroke) chemoattracts circulating stem cells for potential repair. Disclosures: No relevant conflicts of interest to declare.
726 Background: Rhabdomyosracoma (RMS), the most common soft-tissue sarcoma of adolescents and children, frequently infiltrates the BM to such a degree that it often mimics acute lymphoblastic leukemia. The prognosis is poor, particularly for the more aggressive and metastatic alveolar RMS (ARMS) compared to embryonal RMS (ERMS). In our previous work, we demonstrated a pivotal role for two signaling axes, a-chemokine stromal-derived factor-1 (SDF-1)–CXCR4 (a seven-transmembrane-spanning G protein-coupled receptor) and hepatocyte growth factor/scatter factor (HGF/SF)–c-met, in metastasis of pediatric sarcomas to bone marrow (BM) (Blood 2002;100:2597-2606,Cancer Research 2003; 63:7926–7935, IJC 2010;127: 2554–2568). Recently, however, we observed that the bioactive lipids sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) are much more potent chemotractants for human rhabdomyosarcoma (RMS) than SDF-1 or HGF/SF. Importantly, we observed that S1P and C1P levels are highly increased in BM after radio-chemotherapy. Hypothesis: Based on these observations, we hypothesized that S1P and C1P direct chemotaxis of RMS cells to BM. This could be particularly important in patients treated with radio-chemotherapy, where upregulation of S1P and C1P levels in BM may facilitate the spread to the bones of tumor cells that survived initial treatment. Material and Methods: Several complementary in vitro and in vivo approaches were employed to demonstrate a novel role of bioactive lipids in BM metastasis of RMS cells. The expression of S1P seven-transmembrane-spanning G protein-coupled receptors, chemotaxis, adhesion, proliferation, and cell signaling studies in response to S1P and C1P were performed on 8 human ARMS and 3 human ERMS cell lines. The secretion of S1P and C1P in BM and by RMS cells was measured by mass spectrometry (MS). The S1P1 receptor was downregulated by employing an shRNA strategy and S1P1-KO cells were evaluated for their ability to grow tumors in immunodeficient mice. Finally, to address the role of the S1 P–S 1P1 axis in the unwanted spread of sarcoma cells after radio-chemotherapy, we compared seeding of S1P1-KO and control RMS cells in irradiated immunodeficient mice. Results: S1P and C1P are much more potent chemoattractants than SDF-1 or HGF/SF, particularly if employed at “physiological” tissue concentrations. S1P1–5 receptors are expressed on RMS cells and stimulation by S1P induced chemotaxis, adhesion of these cells, and phosphorylation of MAPPp42/44 and AKT. However, while receptor/s for C1P have not yet been identified, C1P also exerted similar effects on human RMS cells. Finally, S1P1-KO cells grew smaller tumors in immunodefcient mice and had impaired seeding efficiency in irradiated animals compared to control RMS cells transduced with empty vector. In parallel experiments, we also observed that both bioactive lipids increase stromalization of the RMS by i) chemoattracting and activating cancer-associated fibroblasts (CAF) and ii) promoting tumor angiogenesis. Conclusions: Both systemic and local radio-chemotherapy leads to upregulation of bioactive lipids in damaged tissues and side effect of such treatment is induction of unwanted prometastatic microenvironment in different organs. By employing an RMS model, we confirmed S1P and identified C1P as novel under-appreciated factor directing metastasis of cancer cells. Since S1P and C1P become upregulated in BM after radio-chemotherapy, both bioactive lipids are involved in the unwanted spread to the bones of RMS cells that survived initial treatment. The role of S1P and C1P in metastasis of other pediatric sarcomas and other types of solid tumors and dissemination of leukemias/lymphomas is currently being investigated in our laboratory, similarly as different strategies to inhibit pro-metastatic effects of S1P and C1P. Disclosures: No relevant conflicts of interest to declare.
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