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HEMATOPOIESIS
From Institut National de la Santé et de la
Recherche Médicale (INSERM) U474, Maternité Port-Royal, and
Laboratoire d'Hématopoïèse, Site Transfusionnel,
Hôpital Cochin, Paris, France; INSERM U448 and Département
de Pathologie EA 2348, Hôpital Mondor, Créteil, France;
IPBS, GDR 1927, Centre National de la Recherche Scientifique UPR 9062, Toulouse, France.
In adult bone marrow, mature erythroblasts are produced
within structures called erythroblastic islands and then cross the endothelial barrier to reach circulation. Erythroblastic islands are
composed of a central macrophage surrounded by maturing erythroblasts. In this study, it is shown that erythroid cells, but not the
other mature hematopoietic cells, coexpress 2 angiogenic factors,
vascular endothelial growth factor A (VEGF-A) and placenta growth
factor (PlGF). Secretion of both VEGF-A and PlGF increases during in vitro erythroid differentiation. Erythroblast-conditioned medium can
induce both migration of monocytes and endothelial cells and the
permeability of endothelial cells. These effects are inhibited by
anti-PlGF and/or anti-VEGF antibodies. Finally, it is shown that VEGF-A
and PlGF proteins are expressed by bone marrow erythroblasts in vivo.
Angiogenic factors secreted by erythroblasts may promote interactions
either with macrophages in erythroblastic islands or with endothelial
cells that would facilitate the passage of erythroid cells through the
endothelial barrier.
(Blood. 2001;97:1968-1974) Erythropoiesis is a multistep process involving the
differentiation of pluripotent hematopoietic stem cells through the
lineage-committed burst-forming unit-erythroid (BFU-E) and
colony-forming unit-erythroid (CFU-E) progenitor cells. These
progenitors give rise to a series of early and late erythroblasts,
leading to the formation of reticulocytes and finally mature
erythrocytes.1 Interactions between hematopoietic precursor cells and their surrounding bone marrow environment are
essential for erythroid differentiation as described in the model of
the Sl/Sld mouse, whose anemia results from bone marrow
stromal defects.2 In the bone marrow of mammals,
erythropoiesis occurs in specific anatomic units called erythroblastic
islands composed of a central macrophage surrounded by a ring of
developing erythroblasts.3-5 Under normal circumstances,
only mature cells cross the endothelial barrier into peripheral blood;
immature cells are retained within the bone marrow.6
Little is known about the initial events leading to the formation of
erythroblastic islands and about interactions between erythroid cells
and endothelial cells.
We have recently reported that neuropilin-1 is expressed on bone marrow
stromal cells and that immature hematopoietic cells express several
neuropilin-1 ligands.7 Neuropilin-1 was first characterized as a receptor for the semaphorin/collapsin protein family, which plays an important role in the guidance of growing axons.8,9 Recently, vascular endothelial growth factor A (VEGF-A)10 and placenta growth factor (PlGF)11
were also identified as neuropilin-1 ligands. VEGF-A is a homodimeric
protein that contains 1 of the 5 VEGF isoforms composed of 121, 145, 165, 189, or 206 amino acids (VEGF-121, VEGF-145, VEGF-165, VEGF 189, or VEGF-206).12 These various isoforms differ in their
affinity for heparin and extracellular matrix components and are
important regulators of angiogenesis and blood vessel
permeability.13 They all bind the tyrosine kinase
receptors, fetal liver kinase 1 (flk-1)14 and fms-like
tyrosine kinase 1 (flt-1).15 Activation of flk-1 is
correlated with the induction of angiogenesis and endothelial cell
proliferation,16,17 and activation of flt-1 enhances
endothelial cell17 and monocyte
migration.18,19 PlGF is a homodimeric protein that shares
substantial structural similarity with VEGF-A.20 Three
PlGF isoforms have been described (PlGF-1, PlGF-2, and
PlGF-3).21,22 PlGF-2 differs from the other 2 as it
contains a heparin-binding domain.23 PlGFs do not
interact with flk-1, but through binding to flt-1,24 they can induce the migration of endothelial cells25 and
monocytes.18,26 Their function in blood vessel
permeability remains uncertain.24,25,27 Finally, PlGF-1
can form active heterodimers with VEGF-A.28
In a search for expression of neuropilin-1 ligands in hematopoietic
cells, we discovered that erythroid cells, but not other mature
hematopoietic cells, produce both VEGF-A and PlGF. We analyzed VEGF-A
and PlGF secretion during in vitro erythroid differentiation and
studied the effects of this production on monocyte chemotaxis and on
endothelial cell migration and permeability. Finally, we showed that
normal bone marrow erythroblasts are a major source of these angiogenic
factors in vivo.
Reverse transcription-polymerase chain reaction
analysis
Cell cultures
For megakaryocytes and granulocytes, CD34+ cells were cultured in serum-free Iscove's Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Cergy Pontoise, France) in the presence of 15% of a commercial mixture of bovine serum albumin, insulin, and transferrin (BIT 9500) (Stem Cell Technologies, Vancouver, BC, Canada). To obtain megakaryocytes, 100 ng/mL thrombopoietin (Kirin Brewery, Tokyo, Japan) and 5 ng/mL stem cell factor (SCF) (Amgen; Thousand Oaks, CA) were added. In these conditions, more than 95% of the cells displayed megakaryocyte-lineage-specific CD41+/CD61+ staining by day 12. To obtain granulocytes, 10 ng/mL SCF, 10 ng/mL interleukin-3, 50 ng/mL flt-3-ligand, and 10 ng/mL granulocyte colony-stimulating factor were added. In these conditions, all the cells displayed a granulocytic phenotype by day 15, as determined both by direct observation after May-Grünwald Giemsa staining and by CD33 immunophenotyping. Human umbilical artery endothelial cells (HUAECs) were cultured in Endothelial SFM Medium (Life Technologies) containing 20% fetal calf serum (FCS) and complemented with 1 ng/mL VEGF every other day. Culture of bovine corneal endothelial (BCE) cells was previously described.30 Enzyme-linked immunosorbent assays Secretion of human VEGF-A (all isoforms) and PlGF (all isoforms) by erythropoietic cells was detected by Quantikine human VEGF and PlGF immunoassays (R&D Systems; Abingdon, United Kingdom) according to the manufacturer's protocol. From day 1 to day 7 of the second stage of culture, erythroid cells were maintained at a concentration of 500 000 cells per milliliter. Every day, 106 cells were harvested and replaced in fresh medium, and collected erythroblast-conditioned medium (ECM) was subjected to a quantitative sandwich immunoassay technique. For PlGF immunoassays, ECM was concentrated 5-fold by means of a Centricon-10 concentrator (Millipore; Bedford, MA).Monocyte migration assay Monocytes were isolated from peripheral blood mononuclear cells of healthy volunteers by means of a monocyte isolation kit (Miltenyi Biotech; Bergish Gladbach, Germany). Monocyte purity evaluated by CD14 staining was greater than 90%. Migration assays were performed by means of the Transwell system (Polylabo; Strasbourg, France).31 The upper and lower wells were separated by a 5-µm pore size polycarbonate filter. In the upper chamber, 2.105 monocytes were placed in a volume of 100 µL. VEGF-16530 and/or PlGF-1 (provided by D. Maglione, Naples, Italy) were added to the lower wells under a final volume of 600 µL in RPMI-1640 plus 1% FCS. ECM was concentrated by means of a Centricon-10 concentrator and added to the lower chamber under a final volume of 600 µL in RPMI-1640 plus 1% FCS. Negative controls were RPMI-1640 plus 1% FCS and cell-free erythroblast culture medium. Blocking rabbit antibodies directed against either human VEGF30 or human PlGF25 were added at a final concentration of 50 µg/mL and 1 µg/mL, respectively. After 5 hours at 37°C, cells present in the lower chamber were numbered.For control analysis, ECM was added in the lower compartment and/or in the upper compartment. ECM a contained 50 pg/mL PlGF and 1.2 µg/mL VEGF-A (30 pg PlGF and 700 pg VEGF-A in 600 µL), and ECM f contained 750 pg/mL PlGF and 18 µg/mL VEGF-A. Endothelial cell migration and permeability assays Migration wound assays were performed on HUAECs as previously described.30 Paracellular permeability assays were performed by measuring tritiated inulin (Amersham, Orsay, France) efflux through BCE cell monolayers (Ortéga et al, manuscript in preparation). Briefly, 105 BCE cells were seeded on 1-cm2 gelatinized filters mounted in diffusion chambers (Nunc, Roskilde, Denmark) in DMEM (Life Technologies) plus 10% FCS. At 4 to 6 days after confluence, cell monolayers were transferred into serum-free medium, and the modulators were added in the upper tank under a final volume of 500 µL in the presence of 2 µCi tritiated inulin. Aliquots of 10 µL were collected from lower chambers at different time intervals during a period of 120 minutes and counted in a scintillation counter. Blocking rabbit antibodies directed against human VEGF30 and against human PlGF25 were added at a final concentration of 50 µg/mL and 1 µg/mL, respectively. Negative controls consisted of cell-free erythroblast culture medium in either the absence or presence of 50 µg/mL rabbit preimmune serum.30Immunohistochemistry We examined 7 bone marrow biopsy samples with normal histological features. The bone marrow biopsies had been performed in the clinical staging of lymphomas. Bone marrow specimens were fixed in Bouin's fixative and then paraffin-embedded. Paraffin-embedded bone marrow sections were evaluated for VEGF-A, PlGF, and CD68 antigens by means of the alkaline phosphatase anti-alkaline phosphatase (APAAP) method.32 For better detection, sections were pretreated with microwave oven heating (3 cycles of 750 W for 5 minutes in 0.01 M citrate buffer, pH 6). Dilutions were 1:50 for anti-VEGF-A (rabbit anti-human VEGF-A [A-20]-G) (Santa Cruz Biotechnology; Santa Cruz, CA); 10 µg/mL for anti-PlGF (rabbit anti-human PlGF-125); and 1:75 for anti-CD68 (PG-M1) (Dako, Trappes, France). For detection of polyclonal antibodies, an incubation step with a mouse antirabbit immunoglobulin was added before proceeding with the APAAP technique. Rabbit antimouse immunoglobulins and APAAP complexes were obtained from Dako. Controls included the omission of the primary antibody and the use of an irrelevant antithyroglobuline polyclonal antibody (Dako). We used 2 combinations of antibodies in the double immuno-enzymatic staining procedure33: VEGF-A/CD68 and PlGF/CD68. Nuclei were counterstained with hematoxylin.To evaluate the coexpression of glycophorin-A and VEGF-A or PlGF in erythroblasts, a double immunofluorescence method34 was used on paraffin-embedded bone marrow sections. VEGF-A and PlGF antibody dilutions were the same as used in APAAP method. Anti-glycophorin-A (Dako) was diluted 1:100. The secondary antibodies, goat F(ab')2 immunoglobulin (Ig)-G (heavy [H] and light [L]) fluorescein isothiocyanate conjugate and goat anti-mouse IgG (H and L) Cy3 were incubated with tissue sections at final concentrations of 20 and 4 µg per milliliter, respectively. Controls included the omission of the primary antibody. Tissue sections were analyzed under a microscope (Leica, Rueil-Malmaison, France) equipped with an epifluorescent system for the detection of fluorescein isothiocyanate, Texas red, or a combination of both.
VEGF-A and PlGF transcripts are coexpressed during in vitro erythroid differentiation We had previously shown that neuropilin-1 was expressed on bone marrow stromal cells and that neuropilin-1 ligand messenger RNAs (mRNAs) were expressed by immature hematopoietic cells.7 In this study, we investigated whether mature hematopoietic cells expressed ligands for neuropilin-1 by RT-PCR analysis. Pure populations of human erythrocytes, granulocytes, or megakaryocytes were obtained after differentiation of CD34+ cells (see "Materials and methods"). VEGF-A transcripts were detected in the 3 hematopoietic cell types (Figure 1A, VEGF-A), but interestingly PlGF mRNAs (PlGF-1 and PlGF-2) were found only in erythroblasts (Figure 1A, PlGF). We therefore focused our attention on erythroid differentiation. Mature erythroblasts expressed 3 VEGF-A mRNAs encoding the VEGF-121, VEGF-145, and VEGF-165 isoforms (Figure 1A, VEGF-A). No expression of the VEGF-189 and VEGF-206 isoforms could be detected. To confirm these results, we performed RNase protection assays. In these experiments, VEGF-145 mRNA was weakly expressed: VEGF-165 mRNA level was 3 times higher than VEGF-145 mRNA, and VEGF-121 mRNA level was 1.5 times higher than VEGF-165 mRNA (data not shown).
During erythroid differentiation, the amounts of the 2 major VEGF-A mRNAs, ie, VEGF-121 and VEGF-165, were roughly invariant from day 1 (corresponding to a majority of CFU-E and late BFU-E progenitors) to day 7 (corresponding to erythroblasts) of the second stage of culture (Figure 1B, VEGF). The major PlGF mRNA, ie, PlGF-1, was detected from day 2 (corresponding to a majority of CFU-E progenitors), and its level gradually increased during erythroid differentiation (Figure 1B, PlGF-1). VEGF-A and PlGF proteins are both secreted by erythroblasts during in vitro differentiation As VEGF-A and PlGF transcripts were detected in erythroblasts, we examined whether these factors were secreted during in vitro erythroid differentiation. From days 1 to 7 of culture, cells were harvested every day and replaced in fresh culture medium. The concentration of VEGF-A and PlGF in collected ECM was evaluated by means of enzyme-linked immunosorbent assays (ELISAs) that take all VEGF-A and PlGF isoforms into account. As PlGF levels in ECM were close to the minimum detectable dose, we measured PlGF levels in 5-fold concentrated ECM. Culture supernatants contained both VEGF-A and PlGF (Figure 2). Secretion of these factors gradually increased during erythroid differentiation by 3-fold for VEGF-A and 100-fold for PlGF (Figure 2). VEGF-A and PlGF levels reached a plateau at day 6 of the second stage of culture (corresponding to differentiated erythroblasts) at 450 pg/mL per 106 cells for VEGF-A and 18 pg/mL per 106 cells (90 pg/mL in 5-fold concentrated ECM as indicated in Figure 2) for PlGF.
ECM induces migration of human monocytes PlGF or VEGF-A receptors (flt-1, flk-1, and neuropilin-1) are not expressed in erythroblastic cells (data not shown). Therefore, we assumed that PlGF and VEGF-A may exclusively act as paracrine effectors. Macrophages are found in close association with erythroid cells within erythroblastic islands, and like their precursors, monocytes, they express the flt-1 receptor. Morever, monocytes are known to migrate in response to PlGF and VEGF-A. Therefore, these cells may constitute potential targets for erythroblasts. To test this hypothesis, we investigated whether ECM was able to induce migration of human monocytes. Monocyte migration was assayed across polycarbonate filters facing ECM, which was collected at day 6 of the second stage of culture. Different ECM volumes (a = 0.6 mL, b = 2 mL, c = 3 mL, d = 4 mL, e = 6 mL, f = 9 mL) were concentrated and used under a final volume of 600 µL so that concentration factors were 1 for a, 3.3 for b, 5 for c, 6.6 for d, 10 for e, and 15 for f. Using increasing ECM volumes, we showed that ECM induced monocyte migration and that the number of migrated cells was directly correlated with PlGF and VEGF-A quantities (Figure 3A). In addition and as shown in Table 1, migration of monocytes depended on the presence of an ECM-positive gradient between the lower and the upper compartments (higher concentration below the filter). Checkerboard analysis has previously revealed that the response of monocytes to VEGF and probably to PlGF was a result of chemotaxis and not of chemokinesis.18 These data and ours indicate that ECM is able to induce a chemotactic response in human monocytes with no appreciable chemokinetic response.
Then we compared the chemotactic activity of ECM with that of recombinant factors. As shown in Figure 3B, 10 ng recombinant PlGF-1 plus 10 ng VEGF-165 induced the migration of 10.5% of monocytes (21 000 migrated cells per well). Similar chemotactic activity (26 000 migrated cells per well) was obtained with ECM a, which contained only 30 pg PlGF and 700 pg VEGF-A. Furthermore, 95% of monocytes migrated (190 000 migrated cells per well) in response to ECM f, which contained 450 pg PlGF and 10 ng VEGF-A. Interestingly, such a migration rate was never reached with the tripeptide fMLP control chemoattractant18 or with higher quantities of recombinant VEGF-165 and/or PlGF-1. Finally, we evaluated the role of VEGF-A and PlGF present in ECM using blocking antibodies. As shown in Figure 3B, chemotaxis induced by ECM f (190 000 migrated monocytes) was inhibited 40% by blocking antibodies against PlGF (114 000 cells, lane ECM + Ab PlGF) and 50% by blocking antibodies against VEGF (95 000 cells, lane ECM + Ab VEGF). Chemotaxis of monocytes was inhibited 70% by both blocking antibodies (57 000 cells, ECM + Ab PlGF + Ab VEGF). Taken together, these data demonstrate that ECM induces a chemotactic response of monocytes and that this activity is mediated in part by PlGF and VEGF-A. In addition, ECM is more potent than recombinant VEGF-A and PlGF. ECM induces endothelial cell migration and permeability Since vascular endothelial cells express VEGF-A and PlGF receptors and interact with erythroid cells in bone marrow, they could also be target cells for VEGF-A and PlGF contained in ECM. Therefore, we investigated whether ECM could induce migration and permeabilization of endothelial cells. In these assays, ECM contained 38 pg/mL VEGF-A and 7 pg/mL PlGF. Figure 4A shows results of migration wound assays performed on HUAECs. ECM induced a significant migration (150 migrated cells per field), which was similar to the results obtained with either 50 ng/mL VEGF-165 (140 migrated cells per field) or 50 ng/mL PlGF-1 (115 migrated cells per field). This migration was inhibited 55% by anti-VEGF blocking antibodies (Figure 4A, ECM + Ab VEGF) or by anti-PlGF blocking antibodies (Figure 4A, ECM + Ab PlGF). Addition of both blocking antibodies totally inhibited this migration (Figure 4A, ECM + Ab VEGF + Ab PlGF).
Permeability assays were performed on BCE monolayers. As shown in Figure 4B, ECM induced inulin efflux through BCE monolayers. The profile of this efflux was similar to that obtained with 50 ng/mL VEGF-165. Activities of ECM and recombinant VEGF-165 were both inhibited in the presence of anti-VEGF blocking antibodies. Anti-PlGF blocking antibodies had no significant effect (data not shown). From these results, we conclude that ECM is able to induce migration of endothelial cells and that this activity is mediated by PlGF and VEGF-A. We also demonstrate that VEGF-A produced by erythroblasts is able to enhance cell permeability. In both experiments, ECM is more potent than recombinant VEGF-A and PlGF. Normal human bone marrow erythroblasts express VEGF-A and PlGF As VEGF-A and PlGF are expressed by erythroblasts in culture, we analyzed the expression of VEGF-A and PlGF in normal human bone marrow by immunohistochemistry. By means of the APAAP method on paraffin-embedded bone marrow tissue sections, many erythroblasts grouped in islands were shown to stain strongly with VEGF-A antibody (Figure 5A). As previously reported,35,36 we could also observe that megakaryocytes and myeloid cells disclosed cytoplasmic staining (data not shown). Using a double immunofluorescence method performed with VEGF-A antibody (Figure 5B) and a specific erythroblastic marker, the glycophorin-A antibody (Figure 5C), we confirmed that most glycophorin-A-positive cells expressed VEGF-A (Figure 5D). Using the APAAP method, we also observed that many erythroid cells grouped in islands displayed a PlGF cytoplasmic staining weaker than VEGF-A staining (Figure 5E). No staining was observed in megakaryocytes and granulocytes (data not shown). A double immunofluorescence technique showed that most PlGF-positive cells (Figure 5F) were also stained with glycophorin-A antibody (Figure 5G), thus confirming the erythroid nature of the PlGF-expressing cells (Figure 5H).
Most of the VEGF-positive erythroblasts were grouped in islands around CD68+ macrophages, as shown by double VEGF-A/CD68 immuno-enzymatic staining (Figure 5I). VEGF-A-positive erythroid cells were also found in the vicinity of sinusoids (Figure 5J). Similar results were obtained by using PlGF antibody (data not shown). These results demonstrate that erythroblastic cells express both VEGF-A and PlGF in normal human bone marrow.
In this study, we show that 2 angiogenic factors, VEGF-A and PlGF, are secreted by human erythroid cells during in vitro differentiation and in vivo in normal human bone marrow. Few data about factors secreted by erythroid cells have been reported so far. Early erythroid progenitors BFU-Es produce Epo.37,38 Rauscher murine erythroleukemia cells and splenic erythroid cells of phenylhydrazine-treated mice produce platelet-derived growth factor.39,40 In this study, we detected VEGF-A expression in erythroblasts, megakaryocytes, and granulocytes; PlGF was expressed only in erythroblasts. Our results are in accordance with previous reports that described VEGF-A expression in normal human hematopoietic cells.41 On the other hand, PlGF was found to be expressed in placenta, cultured endothelial cells, and thyroid,20,23,42,43 but no data about its expression in hematopoietic cells have been reported so far. Interestingly, erythroblasts express no receptor for PlGF and VEGF-A (flt-1, flk-1, or neuropilin-1). This suggests that VEGF-A and PlGF may act as paracrine effectors within the bone marrow. Monocytes and endothelial cells that express VEGF-A and PlGF receptors may be target cells. The ability of ECM to induce migration of human monocytes was tested in a Boyden chamber assay. We show that ECM is able to induce chemotaxis of monocytes in a dose-dependent manner and that the maximal response is obtained with a sample that contains 10 ng VEGF-A and 450 pg PlGF. In addition, we show that monocyte chemotaxis is mediated by VEGF-A and PlGF because both blocking antibodies inhibited it by 70%. Whether VEGF-A and PlGF are sufficient to mediate this function is not clear. We show that 10 ng recombinant VEGF-165 plus 10 ng recombinant PlGF-1 actually induce the same response as an ECM sample containing only 700 pg VEGF-A and 30 pg PlGF. These results clearly indicate that ECM is more potent than recombinant factors and that there may be another component in ECM that is needed for enhancing VEGF-A and PlGF activities. We also demonstrate that ECM is able to induce endothelial cell migration in wound assays. This activity is inhibited 55% by anti-PlGF or by anti-VEGF-A antibodies and is totally inhibited by both antibodies. These data suggest that endothelial cell migration induced by ECM is mediated by PlGF and VEGF-A. Again, ECM is more potent than recombinant factors. Finally, we demonstrate that ECM induces the permeability of a BCE cell monolayer. As this activity is inhibited by anti-VEGF-A antibodies but not by anti-PlGF antibodies, we conclude that permeability may be relayed by VEGF-A. It is interesting to note that in addition to erythrocytes, granulocytes and megacaryocytes also express VEGF-A transcripts and are able to cross the bone marrow endothelial barrier. Therefore, VEGF-A may be the mediator of transendothelial migration of hematopoietic cells through the bone marrow barrier. The roles of different VEGF-A isoforms in mediating VEGF-A activities are poorly understood. In ECM, the major 2 VEGF-A transcripts are VEGF-121 and VEGF-165; VEGF-145 is weakly expressed. Unfortunately, specific reagents to differentiate the relative amounts of the different protein isoforms are not available. Monocytes express the flt-1 receptor, but do not express either the KDR or the neuropilin-1 receptor. In addition, VEGF-121 and VEGF-165, but not VEGF-145, can bind to flt-1. Finally, VEGF-121 and VEGF-165 are chemotactic at equivalent doses for human monocytes, and their properties are mediated by the flt-1 receptor.18,19 Therefore, VEGF-121 and VEGF-165 present in ECM may be equivalent for inducing chemotaxis of monocytes. The roles of VEGF-121, VEGF-165, and VEGF-145 in inducing migration and permeability of endothelial cells remain difficult to define. Neuropilin-1 binding is restricted to VEGF-165, and KDR chemotactic activity is known to be enhanced in the presence of neuropilin-1 by 4 times.10 Therefore, presence of both neuropilin-1 and KDR receptors on endothelial cells may relay VEGF-165 activity on these cells. In conclusion, we show that bone marrow erythroblasts are a source of VEGF-A and PlGF, which may act as paracrine factors on monocytes/macrophages and/or endothelial cells. Our results suggest that PlGF and VEGF-A secreted by erythroblasts may be mediators of a cross-talk between erythroblasts and macrophages to organize or maintain the structure of the erythroblastic island. In addition, our data suggest that angiogenic factors may induce interactions between erythroblasts and endothelial cells that may help mature erythrocytes to cross the bone marrow barrier and reach the peripheral blood.
We thank Drs J. Alain, A. Angelillo-Scherrer, M. L. Boulland, L. Croisille, J. M. Freyssinier, H. Jouault, C. Lacombe, J. Marquet, N. Martin-Garcia, S. Montrereau, and P. Rincé for their advice.
Submitted June 16, 2000; accepted November 17, 2000.
Supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), the Centre National de la Recherche Scientifique (CNRS), and the Ligue contre le Cancer and the Association pour la Recherche contre le Cancer (ARC grant 5715 to V.L.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Valérie Lemarchandel, INSERM U474, Maternité Port-Royal, 123 Bd de Port-Royal, 75014 Paris, France; e-mail: lemarchandel{at}cochin.inserm.fr.
1.
Gregory C, Eaves A.
Three stages of erythropoietic progenitor cell differentiation distinguished by a number of physical and biologic properties.
Blood.
1978;51:527-537 2. Morrison-Graham K, Weston JA. Mouse mutants provide new insights into the role of extracellular matrix in cell migration and differentiation. Trends Genet. 1989;5:116-121[CrossRef][Medline] [Order article via Infotrieve]. 3. Bessis M. L'ilôt erythroblastique, unité fonctionnelle de la moelle osseuse. Rev Hematol. 1958;13:8. 4. Bernard J. The erythroblastic island: past and future. Blood Cells. 1991;17:5-14[Medline] [Order article via Infotrieve]. 5. Mel HC. Theoretical considerations on cell ensembles. Blood Cells. 1991;17:15-25[Medline] [Order article via Infotrieve](discussion 25-27). 6. Petrides P, Dittmann KH. How do normal and leukemic white blood cells egress from the bone marrow? morphological facts and biochemical riddles. Blut. 1990;61:3-13[CrossRef][Medline] [Order article via Infotrieve].
7.
Tordjman R, Ortega N, Coulombel L, Plouet J, Romeo P, Lemarchandel V.
Neuropilin-1 is expressed on bone marrow stromal cells: a novel interaction with hematopoietic cells?
Blood.
1999;94:2301-2309 8. Kolodkin AL, Levengood DV, Rowe EG, Tai YT, Giger RJ, Ginty DD. Neuropilin is a semaphorin III receptor. Cell. 1997;90:753-762[CrossRef][Medline] [Order article via Infotrieve]. 9. He Z, Tessier-Lavigne M. Neuropilin is a receptor for the axonal chemorepellent semaphorin III. Cell. 1997;90:739-751[CrossRef][Medline] [Order article via Infotrieve]. 10. Soker S, Takashima S, Miao HQ, Neufeld G, Klagsbrun M. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell. 1998;92:735-745[CrossRef][Medline] [Order article via Infotrieve].
11.
Migdal M, Huppertz B, Tessler S, et al.
Neuropilin-1 is a placenta growth factor-2 receptor.
J Biol Chem.
1998;273:22272-22278
12.
Ferrara N, Davis-Smyth T.
The biology of vascular endothelial growth factor.
Endocrinol Rev.
1997;18:4-25 13. Neufeld G, Cohen T, Gitay-Goren H, et al. Similarities and differences between the vascular endothelial growth factor (VEGF) splice variants. Cancer Metastasis Rev. 1996;15:153-158[CrossRef][Medline] [Order article via Infotrieve]. 14. Terman BI, Carrion ME, Kovacs E, Rasmussen BA, Eddy RL, Shows TB. Identification of a new endothelial cell growth factor receptor tyrosine kinase. Oncogene. 1991;6:1677-1683[Medline] [Order article via Infotrieve]. 15. Shibuya M, Yamaguchi S, Yamane A, et al. Nucleotide sequence and expression of a novel human receptor-type tyrosine kinase gene (flt) closely related to the fms family. Oncogene. 1990;5:519-524[Medline] [Order article via Infotrieve]. 16. Millauer B, Shawver LK, Plate KH, Risau W, Ullrich A. Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant. Nature. 1994;367:576-579[CrossRef][Medline] [Order article via Infotrieve].
17.
Waltenberger J, Claesson-Welsh L, Siegbahn A, Shibuya M, Heldin CH.
Different signal transduction properties of KDR and Flt-1, two receptors for vascular endothelial growth factor.
J Biol Chem.
1994;269:26988-26995
18.
Barleon B, Sozzani S, Zhou D, Weich HA, Mantovani A, Marmé D.
Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1.
Blood.
1996;87:3336-3343
19.
Clauss M, Weich H, Breier G, et al.
The vascular endothelial growth factor receptor Flt-1 mediates biological activities: implications for a functional role of placenta growth factor in monocyte activation and chemotaxis.
J Biol Chem.
1996;271:17629-17634
20.
Maglione D, Guerriero V, Viglietto G, Delli-Bovi P, Persico MG.
Isolation of a human placenta cDNA coding for a protein related to the vascular permeability factor.
Proc Natl Acad Sci U S A.
1991;88:9267-9271
21.
Cao Y, Chen H, Zhou L, et al.
Heterodimers of placenta growth factor/vascular endothelial growth factor: endothelial activity, tumor cell expression, and high affinity binding to Flk-1/KDR.
J Biol Chem.
1996;271:3154-3162 22. Cao Y, Ji WR, Qi P, Rosin A, Cao Y. Placenta growth factor: identification and characterization of a novel isoform generated by RNA alternative splicing. Biochem Biophys Res Commun. 1997;235:493-498[CrossRef][Medline] [Order article via Infotrieve]. 23. Hauser S, Weich HA. A heparin-binding form of placenta growth factor (PlGF-2) is expressed in human umbilical vein endothelial cells and in placenta. Growth Factors. 1993;9:259-268[Medline] [Order article via Infotrieve].
24.
Park JE, Chen HH, Winer J, Houck KA, Ferrara N.
Placenta growth factor: potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1 but not to Flk-1/KDR.
J Biol Chem.
1994;269:25646-25654 25. Ziche M, Maglione D, Ribatti D, et al. Placenta growth factor-1 is chemotactic, mitogenic and angiogenic. Lab Invest. 1997;76:517-531[Medline] [Order article via Infotrieve].
26.
Hiratsuka S, Minowa O, Kuno J, Noda T, Shibuya M.
Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice.
Proc Natl Acad Sci U S A.
1998;95:9349-9354 27. Sawano A, Takahashi T, Yamaguchi S, Aonuma M, Shibuya M. Flt-1 but not KDR/Flk-1 tyrosine kinase is a receptor for placenta growth factor, which is related to vascular endothelial growth factor. Cell Growth Differ. 1996;7:213-221[Abstract].
28.
DiSalvo J, Bayne ML, Conn G, et al.
Purification and characterization of a naturally occurring vascular endothelial growth factor.placenta growth factor heterodimer.
J Biol Chem.
1995;270:7717-7723 29. Freyssinier JM, Lecoq-Lafon C, Amsellem S, et al. Purification, amplification and characterization of a population of human erythroid progenitors. Br J Haematol. 1999;106:912-922[CrossRef][Medline] [Order article via Infotrieve].
30.
Jonca F, Ortéga N, Gleizes PE, Bertrand N, Plouët J.
Cell release of bioactive fibroblast growth factor 2 by exon 6-encoded sequence of vascular endothelial growth factor.
J Biol Chem.
1997;272:24203-24209 31. Lin CL, Suri RM, Rahdon RA, Austyn JM, Roake JA. Dendritic cell chemotaxis and transendothelial migration are induced by distinct chemokines and are regulated on maturation. Eur J Immunol. 1998;28:4114-4122[CrossRef][Medline] [Order article via Infotrieve]. 32. Cordell J, Falini B, Erber W. Immunoenzymatic labeling of antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J Histochem Cytochem. 1984;32:219-229[Abstract]. 33. Lan HY, Mu W, Nikolic-Paterson DJ, Atkins RC. A novel, simple, reliable, and sensitive method for multiple immunoenzyme staining: use of microwave oven heating to block antibody crossreactivity and retrieve antigens. J Histochem Cytochem. 1995;43:97-102[Abstract]. 34. Liebowitz D. Epstein-Barr virus and a cellular signaling pathway in lymphomas from immunosuppressed patients. N Engl J Med. 1998;20:1413-1421.
35.
Mohle R, Green D, Moore M, Nachman R, Rafii S.
Constitutive production and thrombin-induced release of vascular endothelial growth by human megacaryocytes and platelets.
Proc Natl Acad Sci U S A.
1997;94:663-668 36. Banks R, Forbes M, Kinsey S, et al. Release of the angiogenic cytokine vascular endothelial growth factor (VEGF) from platelets: significance for VEGF measurements and cancer biology. Br J Cancer. 1998;77:956-984[Medline] [Order article via Infotrieve].
37.
Hermine O, Beru N, Pech N, Goldwasser E.
An autocrine role for erythropoietin in mouse hematopoietic cell differentiation [published erratum appears in Blood. 1992;79:3397].
Blood.
1991;78:2253-2260
38.
Stopka T, Zivny JH, Stopkova P, Prchal JF, Prchal JT.
Human hematopoietic progenitors express erythropoietin.
Blood.
1998;91:3766-3772 39. Sytkowski AJ, O'Hara C, Vanasse G, Armstrong MJ, Kreczko S, Dainiak N. Characterization of biologically active, platelet-derived growth factor-like molecules produced by murine erythroid cells in vitro and in vivo. J Clin Invest. 1990;85:40-46.
40.
Keutzer JC, Sytkowski AJ.
Regulated production of a pleiotropic cytokine-platelet-derived growth factor: by differentiating erythroid cells in vitro and in vivo.
Proc Natl Acad Sci U S A.
1995;92:4967-4971 41. Ratajczak MZ, Ratajczak J, Machalinski B, et al. Role of vascular endothelial growth factor (VEGF) and placenta-derived growth factor (PlGF) in regulating human haemopoietic cell growth. Br J Haematol. 1998;103:969-979[CrossRef][Medline] [Order article via Infotrieve]. 42. Maglione D, Guerriero V, Viglietto G, et al. Two alternative mRNAs coding for the angiogenic factor, placenta growth factor (PlGF), are transcribed from a single gene of chromosome 14. Oncogene. 1993;8:925-931[Medline] [Order article via Infotrieve]. 43. Viglietto G, Romano A, Manzo G, et al. Upregulation of the angiogenic factors PlGF, VEGF and their receptors (Flt-1, Flk-1/KDR) by TSH in cultured thyrocytes and in the thyroid gland of thiouracil-fed rats suggest a TSH-dependent paracrine mechanism for goiter hypervascularization. Oncogene. 1997;15:2687-2698[CrossRef][Medline] [Order article via Infotrieve].
© 2001 by The American Society of Hematology.
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  N. Patel, C. S. Gonsalves, M. Yang, P. Malik, and V. K. Kalra Placenta growth factor induces 5-lipoxygenase-activating protein to increase leukotriene formation in sickle cell disease Blood, January 29, 2009; 113(5): 1129 - 1138. [Abstract] [Full Text] [PDF]
|
|
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|
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|
M. J. Telen It really IS the red cell Blood, August 1, 2008; 112(3): 459 - 460. [Full Text] [PDF]
|
|
|
|
|
|
J. A. Chasis and N. Mohandas Erythroblastic islands: niches for erythropoiesis Blood, August 1, 2008; 112(3): 470 - 478. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Patel, C. S. Gonsalves, P. Malik, and V. K. Kalra Placenta growth factor augments endothelin-1 and endothelin-B receptor expression via hypoxia-inducible factor-1{alpha} Blood, August 1, 2008; 112(3): 856 - 865. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. He, H. Hu, R. Braren, S.-Y. Fong, A. Trumpp, T. R. Carlson, and R. A. Wang c-myc in the hematopoietic lineage is crucial for its angiogenic function in the mouse embryo Development, July 15, 2008; 135(14): 2467 - 2477. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Basu, T. K. Lung, W. Lemsaddek, T. G. Sargent, D. C. Williams Jr, M. Basu, L. C. Redmond, J. B Lingrel, J. L. Haar, and J. A. Lloyd EKLF and KLF2 have compensatory roles in embryonic {beta}-globin gene expression and primitive erythropoiesis Blood, November 1, 2007; 110(9): 3417 - 3425. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Christov, F. Chretien, R. Abou-Khalil, G. Bassez, G. Vallet, F.-J. Authier, Y. Bassaglia, V. Shinin, S. Tajbakhsh, B. Chazaud, et al. Muscle Satellite Cells and Endothelial Cells: Close Neighbors and Privileged Partners Mol. Biol. Cell, April 1, 2007; 18(4): 1397 - 1409. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Sasaki, T. Inoguchi, K. Muta, Y. Abe, M. Zhang, K. Hiasa, K. Egashira, N. Sonoda, K. Kobayashi, R. Takayanagi, et al. Therapeutic angiogenesis by ex vivo expanded erythroid progenitor cells Am J Physiol Heart Circ Physiol, January 1, 2007; 292(1): H657 - H665. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Hofmann, K. C. Wollert, G. P. Meyer, A. Menke, L. Arseniev, B. Hertenstein, A. Ganser, W. H. Knapp, and H. Drexler Monitoring of Bone Marrow Cell Homing Into the Infarcted Human Myocardium Circulation, May 3, 2005; 111(17): 2198 - 2202. [Abstract] [Full Text] [PDF]
|
|
|
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|
|
C. Menier, M. Rabreau, J.-C. Challier, M. Le Discorde, E. D. Carosella, and N. Rouas-Freiss Erythroblasts secrete the nonclassical HLA-G molecule from primitive to definitive hematopoiesis Blood, November 15, 2004; 104(10): 3153 - 3160. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Carotta, S. Pilat, A. Mairhofer, U. Schmidt, H. Dolznig, P. Steinlein, and H. Beug Directed differentiation and mass cultivation of pure erythroid progenitors from mouse embryonic stem cells Blood, September 15, 2004; 104(6): 1873 - 1880. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Adams, K. Lenk, A. Linke, D. Lenz, S. Erbs, M. Sandri, A. Tarnok, S. Gielen, F. Emmrich, G. Schuler, et al. Increase of Circulating Endothelial Progenitor Cells in Patients with Coronary Artery Disease After Exercise-Induced Ischemia Arterioscler. Thromb. Vasc. Biol., April 1, 2004; 24(4): 684 - 690. [Abstract] [Full Text] [PDF]
|
|
|
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|
N. Perelman, S. K. Selvaraj, S. Batra, L. R. Luck, A. Erdreich-Epstein, T. D. Coates, V. K. Kalra, and P. Malik Placenta growth factor activates monocytes and correlates with sickle cell disease severity Blood, August 15, 2003; 102(4): 1506 - 1514. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Selvaraj, R. K. Giri, N. Perelman, C. Johnson, P. Malik, and V. K. Kalra Mechanism of monocyte activation and expression of proinflammatory cytochemokines by placenta growth factor Blood, August 15, 2003; 102(4): 1515 - 1524. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. S. Torry, D. Mukherjea, J. Arroyo, and R. J. Torry Expression and Function of Placenta Growth Factor: Implications for Abnormal Placentation Reproductive Sciences, May 1, 2003; 10(4): 178 - 188. [Abstract] [PDF]
|
|
|
|
|
|
Y. Yamada, Y. Oike, H. Ogawa, Y. Ito, H. Fujisawa, T. Suda, and N. Takakura Neuropilin-1 on hematopoietic cells as a source of vascular development Blood, March 1, 2003; 101(5): 1801 - 1809. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Casella, T. Feccia, C. Chelucci, P. Samoggia, G. Castelli, R. Guerriero, I. Parolini, E. Petrucci, E. Pelosi, O. Morsilli, et al. Autocrine-paracrine VEGF loops potentiate the maturation of megakaryocytic precursors through Flt1 receptor Blood, February 15, 2003; 101(4): 1316 - 1323. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. GREUB and D. RAOULT Bartonella: new explanations for old diseases J. Med. Microbiol., November 1, 2002; 51(11): 915 - 923. [Full Text] [PDF]
|
|
|
|
 |
|
 L. Levy, C. Neuveut, C.-A. Renard, P. Charneau, S. Branchereau, F. Gauthier, J. T. Van Nhieu, D. Cherqui, A.-F. Petit-Bertron, D. Mathieu, et al. Transcriptional Activation of Interleukin-8 by beta -Catenin-Tcf4 J. Biol. Chem., October 25, 2002; 277(44): 42386 - 42393. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Wang, N. Miura, A. Bonelli, P. Mole, N. Carlesso, D. P. Olson, and D. T. Scadden Receptor tyrosine kinase, EphB4 (HTK), accelerates differentiation of select human hematopoietic cells Blood, April 15, 2002; 99(8): 2740 - 2747. [Abstract] [Full Text] [PDF]
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 R. J. Klasa, A. F. List, and B. D. Cheson Rational Approaches to Design of Therapeutics Targeting Molecular Markers Hematology, January 1, 2001; 2001(1): 443 - 462. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
) and PlGF (
) contents of ECM were evaluated by means of
ELISAs measuring all VEGF-A and PlGF isoforms. ECM was collected from
106 cells at days 1 to 7 of second stage of culture. For
PlGF, values correspond to protein levels measured in 5-fold
concentrated ECM. Each point represents the mean concentration (pg/mL)
and standard deviation of 4 independent assays.
1
(
) in the different ECMs. Number of migrated cells
(
) or in the presence (
) of blocking anti-VEGF
antibodies and with ECM in the absence (