Incorporation of CXCR4 into membrane lipid rafts primes homing-related responses of hematopoietic stem/progenitor cells to an SDF-1 gradient

Blood ◽  
2005 ◽  
Vol 105 (1) ◽  
pp. 40-48 ◽  
Author(s):  
Marcin Wysoczynski ◽  
Ryan Reca ◽  
Janina Ratajczak ◽  
Magda Kucia ◽  
Neeta Shirvaikar ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Lipid Rafts ◽  
Adhesion Molecule ◽  
Actin Polymerization ◽  
Membrane Lipid ◽  
Small Gtpase ◽  
Calcium Flux ◽  
Urokinase Plasminogen Activator Receptor ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract We found that supernatants of leukapheresis products (SLPs) of patients mobilized with granulocyte–colony-stimulating factor (G-CSF) or the various components of SLPs (fibrinogen, fibronectin, soluble vascular cell adhesion molecule-1 [VCAM-1], intercellular adhesion molecule-1 [ICAM-1], and urokinase plasminogen activator receptor [uPAR]) increase the chemotactic responses of hematopoietic stem/progenitor cells (HSPCs) to stromal-derived factor-1 (SDF-1). However, alone they do not chemoattract HSPCs, but they do increase or prime the cells' chemotactic responses to a low or threshold dose of SDF-1. We observed that SLPs increased calcium flux, phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 and AKT, secretion of matrix metalloproteinases, and adhesion to endothelium in CD34+ cells. Furthermore, SLPs increased SDF-dependent actin polymerization and significantly enhanced the homing of human cord blood (CB)– and bone marrow (BM)–derived CD34+ cells in a NOD/SCID mouse transplantation model. Moreover, the sensitization or priming of cell chemotaxis to an SDF-1 gradient was dependent on cholesterol content in the cell membrane and on the incorporation of the SDF-1 binding receptor CXCR4 and the small GTPase Rac-1 into membrane lipid rafts. This colocalization of CXCR4 and Rac-1 in lipid rafts facilitated guanosine triphosphate (GTP) binding/activation of Rac-1. Hence, we postulate that CXCR4 could be primed by various factors related to leukapheresis and mobilization that increase its association with membrane lipid rafts, allowing the HSPCs to better sense the SDF-1 gradient. This may partially explain why HSPCs from mobilized peripheral blood leukapheresis products engraft more quickly in patients than do those from BM or CB. Based on our findings, we suggest that the homing of HSPCs is optimal when CXCR4 is incorporated in membrane lipid rafts and that ex vivo priming of HSPCs with some of the SLP-related molecules before transplantation could increase their engraftment.

scholarly journals R4 Rgs Subfamily Proteins Suppress Engraftment and Regulate SDF-1/CXCR4 Signaling in Human CD34+ Hematopoietic Stem/Progenitor Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1927-1927
Author(s):  
Kathy Chan ◽  
Tsin Sik Wong ◽  
Karen Li ◽  
Xiao-Bing Zhang ◽  
Ronald Wang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Actin Polymerization ◽  
Conflicts Of Interest ◽  
Calcium Flux ◽  
Rgs Proteins ◽  
Hematopoietic Stem ◽  
Cell Functions ◽  
Stat3 Phosphorylation ◽  
Cd34 Cells

Members of the Regulators of G-protein Signaling (RGS) are GTPase-accelerating proteins and have been implicated in SDF-1-directed trafficking of mature hematopoietic cells. However, their roles in hematopoietic stem and progenitor cells (HSPC) remain largely unknown. In this study, we investigated the expression, functions and mechanism of R4 RGS subfamily members on migration and engraftment of human HSPC. Our results demonstrated that cord blood (CB), bone marrow (BM) and mobilized peripheral blood (MPB) CD34+ cells expressed specific RGS mRNAs, of which RGS1, RGS2, RGS13 and RGS16 were significantly upregulated by SDF-1 (1.6-1.9 fold, n=5, P<0.05). In the presence of AMD3100, a CXCR4 inhibitor, the stimulating effects of SDF-1 on RGS expression were completely abolished (n=6). SDF-1-directed functions (chemotaxis, trans-matrigel migration and calcium flux) and signaling (phosphorylation of Akt, ERK and Stat3) were significantly inhibited in RGS1, RGS13 and RGS16-overexpressing CD34+ cells (n=4-6, P<0.05) but not in RGS2-overexpressing cells, whereas actin polymerization, adhesion and colony formation were unaffected by these RGS members. In the NOD/SCID mouse xenotransplantation model, overexpression of RGS1, RGS13 or RGS16 in CD34+ cells did not impact their short-term homing but substantially compromised their long-term engraftment efficiency in bone marrow and spleens of recipient mice by 91.4%, 83.7% and 71.2%, respectively (n= 8-9; P<0.05). Genome-wide expression microarray and qPCR validation identified 32 common differentially expressed genes (1 upregulated and 31 downregulated) in RGS1, RGS13 or RGS16-overexpressing CD34+ cells. Network analysis revealed the potential mechanisms of RGS1, RGS13 and RGS16 downstream of SDF1/CXCR4 and Gαi protein, leading to compromised Akt, ERK and Stat3 phosphorylation and negative regulation of stem cell functions (CCNA1, SPP1, LPAR5, IL1RL1, HPSE), complement activation (C3AR1, C5AR2, C5AR1), proteolysis (TIMP3, MMP14) and cell migration (THBS1, F2RL2, PROS1, CCL1). Our results highlight the unprecedented functions of R4 RGS proteins in HSPC migration and engraftment, and provide the foundation of future design of RGS-targeting strategies to enhance the efficiency of clinical HSPC transplantation. Disclosures No relevant conflicts of interest to declare.

Impaired Engraftment of Hematopoietic Stem/Progenitor Cells (HSPC) in Immunodefcient Mice Supports an Important Role of Complement System for Optimal Homing.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 338-338
Author(s):  
Reca Ryan ◽  
Marcin Wysoczynski ◽  
Janina Ratajczak ◽  
Mariusz Z. Ratajczak
Keyword(s):  
Progenitor Cells ◽  
Lipid Rafts ◽  
Membrane Lipid ◽  
Scid Mice ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cell Counts ◽  
Cd34 Cells

Abstract Recently we demonstrated that conditioning for transplantation (radio-chemotherapy) activates complement (C) in bone marrow (BM) and that the third complement component (C3) cleavage fragments (C3a and desArgC3a) increase responsiveness of hematopoietic stem/progenitor cells (HSPC) to stromal-derived factor (SDF)-1 gradient by enhancing the incorporation of CXCR4 into membrane lipid rafts – what enables its better interaction with small GTPases from the Rho/Rac family (Blood2003, 101, 3784, Blood2005, 105, 40–48). Based on these data we hypothesized that C could affect the homing/engraftment of HSPC. Thus we performed transplant experiments in several strains of immunodeficient animals. First, we noticed that lethally irradiated NOD/SCID mice engrafted worse with wt HSPC as compared to wt animals (~30% decrease in a presence of donor-derived clonogeneic CFU-GM in marrow cavities 24 hrs after transplantation). This impaired engraftment correlated with the lack of C activation in BM after conditioning for transplantation by lethal irradiation. The lack of C activation in NOD/SCID mice after conditioning for transplant could be explained by a lack of IgM antibodies that activate C by classical IgM-dependent pathway. Next, to learn more on the molecular mechanisms of C cascade activation during conditioning for transplantation and the role of the C3a-C3aR axis in engraftment of HSPC we studied engraftment i) of wild type (wt) murine HSPC in immunodeficient mice (C3−/− and C3aR−/−) and ii) murine HSPC derived from C3aR−/− or C3−/− deficient mice in wild type littermates. The engraftment of HSPC was evaluated by i) recovery of peripheral blood cell counts in transplanted animals, ii) number of CFU-S colonies and iii) number of clonogeneic progenitors in marrow cavities 16 day after transplantation. We noticed that both C3−/− and C3aR−/− mice had impaired engraftment with wt HSPC. At the same time HSPC from C3aR−/− mice but not C3−/− animals showed poor engraftment in wt recipients. This suggests that i) C3aR expressed on HSPC interacts with C3a generated during C-activation in BM environment and ii) that this interaction is important for optimal homing of HSPC. To support further this notion, human CD34+ cells were exposed to nontoxic doses of C3aR antagonist SB290157 and transplanted into NOD/SCID mice. Subsequently, 24 hrs after transplantation cells were isolated from the marrow cavities and stimulated to grow human CFU-GM colonies. By employing this assay we noticed reduced engraftment of human CD34+ cells (~30%, p<0.0001) as compared to mice transplanted with control CD34+ cells unexposed to SB290157. These data allow for the following conclusions: i) C is activated in BM during conditioning for transplantation by irradiation ii) C is activated after exposure of a natural neoepitope in damaged marrow which is recognized by natural IgM activating C via the classical pathway, iii) C3 cleavage product C3a binds to C3aR on transplanted HSPC and increases incorporation of CXCR4 into membrane lipid rafts enhancing responsiveness of HSPC to an SDF-1 gradient, and finally iv) a proper interplay between the C system and SDF-1-CXCR axis ensures optimal homing of HSPC.

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

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3536-3536
Author(s):  
Neeta Shirvaikar ◽  
Leah A. Marquez-Curtis ◽  
Andrew Shaw ◽  
A. Robert Turner ◽  
Anna Janowska-Wieczorek
Keyword(s):  
Flow Cytometry ◽  
Steady State ◽  
Western Blot ◽  
Lipid Rafts ◽  
Stromal Cells ◽  
Hematopoietic Cells ◽  
Membrane Lipid ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Cd34 Cells

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

Novel Evidence That Statin-Mediated Perturbation of Lipid Raft Formation Ameliorates Bleeding- Related Thrombocytosis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2164-2164
Author(s):  
Marcin Wysoczynski ◽  
Ryan Reca ◽  
Magda Kucia ◽  
Janina Ratajczak ◽  
Mariusz Z. Ratajczak
Keyword(s):  
Lipid Rafts ◽  
Lipid Raft ◽  
Actin Polymerization ◽  
Membrane Lipid ◽  
Calcium Flux ◽  
Cholesterol Depletion ◽  
Cholesterol Lowering ◽  
Platelet Production ◽  
Cell Counts ◽  
Stress Dependent

Abstract The a-chemokine stromal derived factor 1 (SDF-1) plays an important role in maturation/platelet formation of megakaryocytes (Megs), and we recently reported that the responsiveness of hematopoietic cells to SDF-1 is optimal when its corresponding CXCR4 receptor is included into membrane lipid rafts (Blood2005; 105:40). The formation of lipid rafts in vivo may be perturbed by cholesterol-lowering drugs, e.g., statins. Statins are effective in lowering LDL cholesterol and exert several pleiotropic effects on mature platelets, e.g., inhibit their activation. This latter effect is probably due to lowering of the cholesterol content in the membranes of mature platelets leading to the perturbation of membrane lipid raft formation which is required for proper platelet activation and signaling. However, under steady state conditions statins do not influence platelet production, no studies have been performed on their effects on platelet production in reactive thrombocytosis. In determining whether lipid raft formation plays a role in SDF-1- and stress-dependent thrombocytosis, we found that SDF-1 most efficiently stimulates in vitro platelet production when CXCR4 is included into membrane lipid rafts on Megs. At the molecular level, depletion of cholesterol from Megs membranes i) perturbed the responsiveness of megakaryocytic progenitors to an SDF-1 gradient, ii) inhibited SDF-1-mediated calcium flux and MAPKp42/44, AKT and STAT 1–6 phosphorylation in normal human Megs and (iii) inhibited F-actin polymerization, MMP-9 and VEGF secretion, and adhesion to endothelium and fibrinogen. More importantly we found that ex vivo-expanded human Megs, produced significantly fewer platelets during their transendothelial migration after preincubation with cholesterol-lowering MbCD. To evaluate whether cholesterol depletion from Megs affects platelet production we exposed C57Bl6 mice to statins (orally, 21 days, 750 mg/mouse) and observed that statins did not influence peripheral blood cell counts. However, when mice were acutely bled this treatment led to a significant amelioration of post-bleeding thrombocytosis as compared to untreated (control) mice. At the same time, recovery of erythrocyte and leukocyte counts was unaffected. Thus we demonstrated for the first time that statins affect CXCR4-mediated thrombopoiesis by perturbing lipid raft formation and should be considered as potential drugs to prevent post-trauma or post-operative thrombocytosis.

Upregulation of MT1-MMP by Molecules Present in Leukaphereis Products Primes CD34+ Cell Homing.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5273-5273
Author(s):  
Neeta Shirvaikar ◽  
Jencet Montano ◽  
A.Robert Turner ◽  
Mariusz Z. Ratajczak ◽  
Anna Janowska-Wieczorek
Keyword(s):  
Lipid Rafts ◽  
Leading Edge ◽  
Membrane Lipid ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Cell Homing ◽  
Mmp Inhibitor ◽  
Cd34 Cells ◽  
Mobilized Peripheral Blood ◽  
Stimulation Of

Abstract Hematopoietic stem/progenitor cells (HSPC) derived from mobilized peripheral blood engraft faster than those from bone marrow (BM) or cord blood (CB). We recently postulated that leukapheresis product supernatants (collected from G-CSF-mobilized patients) and their components (fibrinogen, fibronectin, thrombin, hyaluronic acid (HA) and C3 cleavage fragments) have a priming effect on HSPC homing by enhancing chemotactic responses to SDF-1 through increased incorporation of CXCR4 into membrane lipid rafts and stimulation of matrix metalloproteinase (proMMP-2 and proMMP-9) secretion in these cells (Blood2005; 105:40). As MT1-MMP has been shown to activate latent forms of MMP-2 and (indirectly) MMP-9 and is known to localize proteolytic activity at the leading edge of migrating cells, we decided to investigate MT1-MMPs’ role in the priming of HSPC homing. We observed that stimulation of BM and CB CD34+ cells with priming agents (HA, fibrinogen or thrombin) not only increased secretion of proMMP-2 and proMMP-9 (zymography) but also highly upregulated levels of MT1-MMP transcript (RT-PCR) and protein (Western blotting) in these cells. Moreover, trans-Matrigel chemoinvasion of CD34+ cells towards a low SDF-1 gradient (20 ng/mL), enhanced by priming agents, was inhibited by the potent MT1-MMP inhibitor epigallocatechin-3-gallate. Furthermore, priming agents (HA, fibrinogen and thrombin) increased levels of active MMP-2 in co-cultures of stromal cells (endothelial cells and BM fibroblasts) with CD34+ cells. To elucidate the mechanism of MT1-MMP upregulation by priming agents, we evaluated whether they affected MT1-MMP incorporation in ganglioside M1-enriched membrane lipid rafts in the same way as for CXCR4. We found that stimulation of hematopoietic and THP-1 cells with HA, fibrinogen and thrombin increased incorporation of MT1-MMP into membrane lipid rafts and these observations were confirmed using confocal microscopy. Hence it appears that MT1-MMP, like CXCR4, localizes in membrane lipid rafts, and this is enhanced by priming agents, leading to better homing of HSPC.

A Novel Role for Thrombin in Hematopoietic Stem/Progenitor Cell Homing.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3374-3374
Author(s):  
Neeta Shirvaikar ◽  
Ali Jalili ◽  
Mariusz Z. Ratajczak ◽  
Anna Janowska-Wieczorek
Keyword(s):  
Protein Expression ◽  
Lipid Rafts ◽  
Leading Edge ◽  
Membrane Lipid ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
And Migration ◽  
Stimulation Of

Abstract Thrombin, an important serine protease, not only plays a pivotal role in platelet aggregation and coagulation, but also through activation of its receptor, seven transmembrane, G-protein-coupled receptor PAR-1, elicits numerous cellular responses in platelets and endothelial cells such as induction of adhesion molecules, production of chemokines, activation of matrix metalloproteinase (MMP)-2, cytoskeletal reorganization and migration. Thrombin is also one of the inflammatory molecules elevated during G-CSF mobilization of hematopoietic stem/progenitor cells (HSPC) and their collection by leukapheresis. We recently reported that components of leukapheresis products including thrombin enhance in vitro chemotaxis of CD34+ cells towards an SDF-1 gradient and in vivo homing to bone marrow (BM) niches in a murine model (Blood2005; 105:40). In this study we investigated whether thrombin enhances the homing-related responses of human HSPC (CD34+ cells) through MMPs, especially membrane-type (MT)1-MMP which is known to be localized on the leading edge of migrating cells and both activates latent proMMPs (MMP-2, -9) and itself has strong pericellular proteolytic activity. We found that stimulation of CD34+ cells with thrombin upregulates mRNA for MT1-MMP and MMP-9 as well as MT1-MMP protein expression (Western blot, flow cytometry) and proMMP-2 and proMMP-9 secretion (zymography). Thrombin was also found to (i) prime trans-Matrigel chemoinvasion of CD34+ cells towards a low SDF-1 gradient (20 ng/mL), which was inhibited by epigallocatechin-3-gallate, a potent inhibitor of MT1-MMP, and (ii) activate MMP-2 in of co-cultures of CD34+ cells with stromal cells (BM fibroblasts and HUVEC) which secrete proMMP-2. We also found that SDF-1 upregulates mRNA and protein expression of MT1-MMP. Moreover, using confocal microscopy we demonstrate for the first time that in CD34+ cells, PAR-1, like CXCR4, is localized in the GM1 fraction of lipid rafts and stimulation of these cells with thrombin as well as SDF-1 increases incorporation of MT1-MMP into membrane lipid rafts. Furthermore, disruption of lipid raft formation by the cholesterol-depleting agent methyl-b-cyclodextrin inhibits MT1-MMP incorporation into membrane lipid rafts and also trans-Matrigel chemoinvasion of CD34+ cells towards SDF-1. Thus we conclude that thrombin, through PAR-1 signalling and the SDF-1-CXCR4 axis, upregulates the incorporation of MT1-MMP into membrane lipid rafts and the interaction of these axes enhances the homing-related responses of HSPC towards SDF-1.

Fibrinogen Enhances Homing-Related Responses of CD34+ by Incorporating Membrane Type1-Matrix Metalloproteinase into Membrane Lipid Rafts.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3188-3188
Author(s):  
Neeta Shirvaikar ◽  
A. Robert Turner ◽  
Mariusz Z. Ratajczak ◽  
Anna Janowska-Wieczorek
Keyword(s):  
Lipid Rafts ◽  
Stromal Cells ◽  
Leading Edge ◽  
Membrane Lipid ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Chemokine Gradient ◽  
Cord Blood Cells ◽  
First Time ◽  
Mobilized Peripheral Blood

Abstract Clinical studies have shown that transplanted mobilized peripheral blood (mPB) hematopoietic stem/progenitor cells (HSPC) home faster to the bone marrow (BM) than harvested BM or cord blood cells. We previously showed that fibrinogen has a priming effect on HSPC homing by enhancing the chemotactic responses of CD34+ cells towards a low SDF-1 gradient by incorporating CXCR4 into membrane lipid rafts (Blood2005; 105:40). In this study we further investigated the mechanism of priming by determining the effect of fibrinogen on matrix metalloproteinases (MMPs) expressed by CD34+ cells, particularly MT1-MMP which is localized on the leading edge of migrating cells and not only activates latent MMPs but by itself degrades several extracellular matrix (ECM) components such as collagens, laminin, fibrin and fibronectin. We found that fibrinogen highly upregulated MT1-MMP protein expression (Western blot) in CD34+ cells as well as proMMP-2 and proMMP-9 secretion (zymography); primed trans-Matrigel chemoinvasion of CD34+ cells towards a low SDF-1 gradient (20 ng/mL), which was inhibited by epigallocatechin-3-gallate, a potent inhibitor of MT1-MMP; and stimulated MMP-2 activation in co-cultures of stromal cells (BM fibroblasts and HUVEC) with CD34+ cells. Moreover, we demonstrate by confocal microscopy, for the first time, that in CD34+ cells MT1-MMP is localized in the GM1-fraction of lipid rafts where it co-localizes with CXCR4; this co-localization is enhanced when CD34+ cells are stimulated with fibrinogen. Furthermore, disruption of lipid raft formation by the cholesterol-depleting agent methyl-b-cyclodextrin inhibited MT1-MMP incorporation into membrane lipid rafts and also trans-Matrigel chemoinvasion of CD34+ cells towards an SDF-1 gradient. Thus we conclude that fibrinogen enhances homing-related responses of CD34+ cells towards SDF-1 by increased incorporation and co-localization of CXCR4 and MT1-MMP in membrane lipid rafts. Further, we postulate that while the presence of CXCR4 in lipid rafts allows the cells to better sense the SDF-1 chemokine gradient, the upregulated MT1-MMP in the lipid rafts facilitates their migration through the ECM and possibly towards the BM niches.

Novel Evidence That, in Addition to Proteolytic Enzymes, Lipolytic Enzymes Are Involved in Mobilization of Hematopoietic Stem/Progenitor Cells (HSPCs) - an Important Pro-Mobilizing Role Identified for Hematopoietic-Specific Phospholipase C (PLCβ2)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2448-2448 ◽  
Author(s):  
Sylwia Borkowska ◽  
Agata Poniewierska-Baran ◽  
Gabriela Schneider ◽  
Daniel Pedziwiatr ◽  
Malwina Suszynska ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Progenitor Cells ◽  
Lipid Rafts ◽  
Mononuclear Cells ◽  
Proteolytic Enzymes ◽  
Membrane Lipid ◽  
Retention Function ◽  
Gpi Anchor ◽  
Lipolytic Enzymes ◽  
Hematopoietic Stem

Abstract Background . Hematopoietic stem/progenitor cells (HSPCs) express the chemokine receptor CXCR4 and the very late antigen 4 receptor (VLA-4, also known as alpha4beta1 integrin) on their cell surface and are retained in bone marrow (BM) niches by interaction of these receptors with their respective ligands, α-chemokine stromal-derived growth factor 1 (SDF-1) and vascular adhesion molecule 1 (VCAM-1, also known as CD106), which are expressed by cells in the BM microenvironment (e.g., osteoblasts and fibroblasts). Mobilization studies employing small molecule antagonists of CXCR4 or VLA-4 indicate the importance of both axes in retention of HSPCs in the BM microenvironment. Furthermore, it has been postulated that a crucial role in the mobilization process plays activation of a proteolytic microenvironment (J Clin Invest. 2003;111:187-96) and complement cascade (ComC) (Blood 2004;103:2071-8) in the BM microenvironment. However, to our surprise no attention has been paid so far to the role of lipolytic enzymes. Phospholipase C (PLC) is an enzyme released by neutrophils that cleaves the phosphoglycerol bond in glycosylphospahtidylinositol (GPI anchor), a glycolipid that is attached to the C-termini of several important proteins during posttranslational modification. GPI-linked proteins are thought to be preferentially located in lipid rafts. The most important GPI-anchored proteins include VCAM-1, complement inhibitors CD55 and CD59, and uPAR. We have reported that for retention of HSPCs in BM, both CXCR4 and VLA-4 have to be incorporated into membrane lipid rafts (Blood 2005;105:40-48). Hypothesis . Based on our previous observation that the HSPCs of paroxysmal nocturnal hemoglobinuria patients show defective retention in BM due to lack of functional GPI anchor (Leukemia 2012; 26:1722—5) we have hypothesized that PLC in normal BM may facilitate mobilization of HSPCs by perturbing expression of GPI anchor, which is so crucial for proper retention of HSPCs in BM. Materials and Methods . PLC activity has been measured by ELISA in the BM of mobilized mice as well in conditioned media from neutrophils exposed to several pro-mobilizing factors (G-CSF, AMD3100, C3a, C5a, desArgC5a, and S1P). We also tested the effect of PLC on the expression of VCAM-1 on BM-derived stroma and CD55 and CD59 antigens on BM mononuclear cells. The effect of PLC on incorporation of CXCR4 and VLA-4 into membrane lipid rafts has been studied by confocal microscopy employing murine Sca-1+ and human CD34+ cells. Here, to test our hypothesis, mobilization studies using AMD3100 and G-CSF have been performed in PLCβ2-KO (PLCβ2–/–) mice and their wild type (WT) littermates. Results . Our data indicate that the PLC level increases in BM during mobilization and is released from neutrophils in response to several pro-mobilization factors (G-CSF, AMD3100, C3a, C5a, desArgC5a, and S1P). PLC efficiently cleaves VCAM-1 expressed on BM stromal cells and thus perturbs the VCAM-1–VLA4 interaction as well as removes CD55 and CD59 from BM mononuclear cells, which enhances the pro-mobilizing effects of the ComC. PLC also inhibits lipid raft formation on HSPCs and by this means impairs the normal BM-retention function of CXCR4 and VLA-4. Finally, what is most important, we observed a mobilization defect in PLCβ2–/– mice, as evaluated by the number of mobilized leucocytes, SKL cells, and CFU-GM. Conclusions . We have established for the first time that, in addition to proteolytic enzymes, lipolytic enzymes, including LPC, are upregulated in the BM microenvironment, and that PLC promotes mobilization of HSPCs by perturbing the BM-retention function of GPI-anchored proteins. These data support an important role for GPI anchor-dependent proteins in the retention of HSPCs in BM niches. Disclosures No relevant conflicts of interest to declare.

Functional receptor for C3a anaphylatoxin is expressed by normal hematopoietic stem/progenitor cells, and C3a enhances their homing-related responses to SDF-1

Blood ◽  
2003 ◽  
Vol 101 (10) ◽  
pp. 3784-3793 ◽  
Author(s):  
Ryan Reca ◽  
Dimitrios Mastellos ◽  
Marcin Majka ◽  
Leah Marquez ◽  
Janina Ratajczak ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Calcium Flux ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Wide Range ◽  
Normal Human ◽  
Anaphylatoxin C3a

Abstract Complement has recently been implicated in developmental pathways and noninflammatory processes. The expression of various complement components and receptors has been shown in a wide range of circulating myeloid and lymphoid cells, but their role in normal hematopoiesis and stem cell homing has not yet been investigated. We report that normal human CD34+ cells and lineage-differentiated hematopoietic progenitors express the complement anaphylatoxin C3a receptor (C3aR) and respond to C3a. Moreover, C3a, but not the biologically inactive desArg-C3a, induces calcium flux in these cells. Furthermore, we found that C3 is secreted by bone marrow stroma and that, although C3a does not influence directly the proliferation/survival of hematopoietic progenitors, it (1) potentiates the stromal cell–derived factor 1 (SDF-1)–dependent chemotaxis of human CD34+ cells and lineage-committed myeloid, erythroid, and megakaryocytic progenitors; (2) primes SDF-1–dependent trans-Matrigel migration; and (3) stimulates matrix metalloproteinase-9 secretion and very late antigen 4 (VLA-4)–mediated adhesion to vascular cell adhesion molecule 1 (VCAM-1). Furthermore, we found that murine Sca-1+ cells primed by C3a engrafted faster in lethally irradiated animals. These results indicate that normal human hematopoietic stem and progenitor cells express functional C3aR and that the C3aR-C3a axis sensitizes the responses of these cells to SDF-1 and thus may be involved in promoting their homing into the bone marrow via cross talk with the SDF–CXC chemokine receptor-4 (CXCR4) signaling axis. C3a is the first positive regulator of this axis to be identified.

scholarly journals P-Selectin–Coated Microtube for Enrichment of CD34+ Hematopoietic Stem and Progenitor Cells from Human Bone Marrow

2008 ◽  
Vol 54 (1) ◽  
pp. 77-85 ◽  
Author(s):  
Srinivas D Narasipura ◽  
Joel C Wojciechowski ◽  
Nichola Charles ◽  
Jane L Liesveld ◽  
Michael R King
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Adhesion Molecule ◽  
Mononuclear Cells ◽  
Air Embolism ◽  
Cell Capture ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Coated Surfaces

Abstract Background: Enrichment and purification of hematopoietic stem and progenitor cells (HSPCs) is important in transplantation therapies for hematologic disorders and in basic stem cell research. Primitive CD34+ HSPCs have demonstrated stronger rolling adhesion on selectins than mature CD34− mononuclear cells (MNCs). We have exploited this differential rolling behavior to capture and purify HSPCs from bone marrow by perfusing MNCs through selectin-coated microtubes. Methods: Bone marrow MNCs were perfused through the cell-capture microtubes coated with adhesion molecules. We washed the device lumen and visualized and estimated captured cells by video microscopy. Adherent cells were eluted by high shear, calcium-free buffer, and air embolism. We used immunofluorescence staining followed by flow cytometry to analyze CD34+ HSPCs. Results: CD34+ HSPC purity of cells captured in adhesion molecule–coated devices was significantly higher than the fraction of CD34+ cells found in bone marrow MNCs [mean (SE) 2.5% (0.8%)]. P-selectin–coated surfaces yielded 16% to 20% CD34+ cell purity, whereas antibody-coated surfaces yielded 12% to 18%. Although CD34+ cell purity was comparable between selectin and antibody surfaces, the total number of CD34+ HSPCs captured was significantly higher in P-selectin devices (approximately 5.7 × 104 to 7.1 × 104) than antibody devices (approximately 1.74 × 104 to 2.61 × 104). Conclusions: P-selectin can be used in a compact flow device to capture HSPCs. Selectin-mediated capture of CD34+ HSPCs resulted in enrichment approximately 8-fold higher than the CD34+ cell population from bone marrow MNCs. This study supports the hypothesis that flow-based, adhesion molecule–mediated capture may be a viable alternative approach to the capture and purification of HSPCs.

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