From the
Departments of Medicine and **Pathology and Laboratory Medicine, University of
British Columbia, Vancouver, British Columbia V6T 1Z4, Canada and the
Department of Medical Biophysics, the
Department of Pathology and Laboratory Medicine, and ||Terry Fox Laboratories, British Columbia Cancer Agency, Vancouver, British
Columbia V5Z 1L3, Canada
Received for publication, December 1, 2002 , and in revised form, March 28, 2003.
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ABSTRACT |
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INTRODUCTION |
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VEGF and its receptors have also been shown to play an important role in adult hematopoiesis. VEGFR-2 has been found to be expressed on a subset of hematopoietic stem cells that can differentiate into hematopoietic or vascular endothelial cells, depending on the culture conditions (1012). Recent studies have shown that VEGF can recruit both hematopoietic cells (mainly through VEGFR-1) and endothelial progenitors (through VEGFR-2) to distant sites in vivo. This recruitment of marrow precursors may be critical in tumor angiogenesis (13). Furthermore, inhibition of VEGF and/or its receptors has recently been shown to reduce the number of hematopoietic progenitors in vivo (14). However, because of the numerous members of the VEGF ligand and receptor family, it is difficult to study the specific effects of VEGFR-2 signaling without the interference of other VEGF receptors such as VEGFR-1, VEGFR-3 (Flt-4), and the neuropilins (15).
Recently, the unique signaling effects of some hematopoietic receptors (Flt-3, Mpl, granulocyte-colony stimulating factor receptor, c-Kit) have been studied by fusing the signaling domain of these receptors to an FK506-binding protein (FKBP) that can be specifically activated using synthetic FKBP ligands (1622). This system has permitted the demonstration that the self-renewal and differentiation of hematopoietic progenitors can be influenced through distinct, receptor-initiated signaling pathways (23).
In this study, we used this inducible dimerization strategy to specifically study the effects of VEGFR-2 signaling on hematopoietic progenitors. It has been shown that neuropilin-1 is a receptor for VEGF and acts as a co-receptor that enhances the function of VEGF through VEGFR-2 (24). Furthermore, VEGFR-2 has been shown to heterodimerize with VEGFR-1 (25). The strategy we used allows us to study the unique signaling properties of VEGFR-2, without any interference from other VEGF receptors, allowing us to exclude the effects of neuropilin, or heterodimerization with VEGFR-1. To specifically study the unique signaling effects of VEGFR-2, we fused the cytoplasmic domain of this receptor, which contains the split tyrosine kinase domain, to a mutated FKBP12 domain that harbors a phenylalanine to valine mutation at amino acid 36. Although other studies have shown the signaling effects of VEGFR-2 by using VEGFR-2-specific ligands, such as VEGF-E (26), the use of a nontoxic chemical inducer of dimerization, AP20187 (Ariad Pharmaceuticals), allows us to study with high specificity VEGFR-2 signaling pathways in a cell autonomous manner. This strategy also allows us to rule out any potential signaling effects that could be triggered by neuropilin-1, which acts as a co-receptor for VEGF, enhancing its binding to VEGFR-2 (15). Moreover, AP20187 is well tolerated in vivo, which allows its use in studying specific signaling pathways in vivo and evaluation of its potential use in therapeutic strategies. Our studies show that VEGFR-2 activation results in maintenance of the hematopoietic progenitor population in conditions of cytokine starvation. This effect is mainly due to increased survival of hematopoietic progenitors through the PI 3-kinase/Akt pathway, although the Erk1/2 MAP kinase pathway may also be involved. Our results suggest that VEGFR-2 may be important in maintaining hematopoiesis by promoting the survival of hematopoietic progenitors, through the activation of PI 3-kinase, and possibly through Erk1/2 MAP kinases.
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EXPERIMENTAL PROCEDURES |
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Ecotropic packaged virus was generated using the following procedure. Phoenix-AMPHO cells (R. Nolan) were transfected with the vector plasmids using Fugene (Roche Applied Science, Laval, Canada) according to the instructions of the manufacturer. Medium was changed after 24 h, and transfected cells were cultured for another 24 h in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS). Supernatant was then harvested, filtered, and used for repeated infections of GP+E86 ecotropic packaging cells in the presence of 8 µg/ml polybrene (Sigma). After sorting for GFP expression, transduced GP+E86 cells were plated at limiting dilution. Individual clones were tested, and the highest titer clone was selected by titration of supernatants on NIH 3T3 cells.
Cell LinesHMEC-1 endothelial cells (Center for Disease Control and Prevention, Atlanta, GA) were cultured in MCDB medium (Invitrogen) supplemented with 10% FBS and 10 µg/ml epidermal growth factor (EGF) (Sigma). HMEC-1 cells were retrovirally transduced using amphotropic packaged virus obtained by harvesting the supernatant of Phoenix-AMPHO cells transfected with vector plasmids 48 h prior to supernatant collection. Hematopoietic progenitors were extracted from the femurs and tibias of C3Pep mice (cross between C3H/HeJ and Pep3b) treated 4 days previously with 150 mg/kg 5-fluorouracil (Amersham Biosciences) and cultured for 48 h in Iscove's modified Dulbecco's medium (IMDM) supplemented with a serum substitute (BIT (Stem Cell Technologies Ltd., Vancouver, Canada)), 104 M 2-mercaptoethanol, 40 µg/ml low density lipoproteins (Sigma), 1 ng/ml Flt3-ligand, 300 ng/ml stem cell factor (SCF), and 20 ng/ml interleukin-11 (Stem Cell Technologies). After stimulation, cells were harvested and infected by either cocultivation with irradiated (1500 centigrays, x-ray) GP+E86 viral producer cells or by the addition of virus-containing supernatant from the GP+E86 producer cells in fibronectin-coated dishes. Both infection protocols involved 48-h growth on tissue culture plates with the above cytokine combination and with the addition of 5 µg/ml protamine sulfate (Sigma). Following infection, bone marrow cells were plated in the same medium for another 2 days. Cells were then sorted for GFP expression (FACS 440; Becton Dickinson).
Viability AssaysSorted bone marrow cells were plated in IMDM supplemented with 10% FBS with or without the addition of 100 nM AP20187. We found that this dose-induced maximal survival effect on hematopoietic progenitors (data not shown). Cells were harvested at various times and counted on a hemacytometer.
CFC AssayTransduced GFP-positive bone marrow cells were grown in IMDM supplemented with 10% FBS, with or without 100 nM AP20187, for 7 and 14 days. At these time points, hematopoietic clonogenic progenitor frequencies were determined by plating 20,000 bone marrow cells in methylcellulose medium containing 50 ng/ml SCF, 10 ng/ml IL-3, 10 ng/ml IL-6, and 3 units/ml erythropoietin (Methocult GF M3434; Stem Cell Technologies). Resultant colonies were scored after 10 days of incubation.
CFU-Spleen (CFU-S12) AssayTransduced GFP-positive bone marrow cells were cultured in IMDM supplemented with 10% FBS with or without 100 nM AP20187 for 7 days. 25,000 cells were injected in the tail vein of lethally irradiated (900 centigrays, using a 137Cs source) B6C3 mice (cross between C3H/HeJ and C57Bl/6J). 12 days later, mice were sacrificed, spleens were harvested and fixed in Telleyesniczky's solution, and hematopoietic colonies were counted.
ImmunofluorescenceFor BrdUrd staining, sorted GFP-positive bone marrow cells were cultured in IMDM supplemented with 10% FBS for 2 days and then treated for 2 h with 10 µM BrdUrd with or without 100 nM AP20187. Cytospin preparations of bone marrow cells were fixed with 4% paraformaldehyde for 5 min, washed with phosphate-buffered saline, and permeabilized with ice-cold methanol for 1 min. Slides were then incubated for 20 min at 37 °C with 2 N HCl to denature DNA. Slides were blocked in phosphate-buffered saline, 5% goat serum, 0.1% Triton X-100 for 10 min, followed by a 1-h incubation with primary antibody (anti-BrdUrd conjugated with AlexaFluor 594 (Molecular Probes, Inc., Eugene, OR), 1:50 dilution in phosphate-buffered saline, 5% goat serum, 0.1% Triton X-100). After washing, nuclear DNA was stained with 4',6-diamidino-2-phenylindole (1 µg/ml), and slides were mounted in anti-fading solution. For activated caspase 3 staining, cytospin preparations of hematopoietic progenitors grown in culture for 14 days in IMDM containing 10% FBS were stained using the same protocol as above (the DNA denaturation step was omitted), and the following antibodies were used: anti-activated caspase 3 (BD Pharmingen, San Diego, CA) and goat anti-rabbit Ig conjugated with Texas Red (Molecular Probes).
ImmunoblottingProteins from total cellular extracts were separated by SDS-PAGE and assessed by immunoblotting as previously described (29). Antibodies against phosphorylated VEGFR-2 and total and phosphorylated Akt and Erk MAP kinase were obtained from Cell Signaling Technology (Mississauga, Canada). Anti-HA antibody was obtained from Babco (Richmond, CA). The kinase inhibitors LY294002 and U0126 were obtained from Calbiochem.
Statistical AnalysisA two-tailed Student t test was used to determine differences between treated and untreated cultures. p values < 0.05 were considered statistically significant.
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RESULTS |
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To investigate the effect of VEGFR-2 in hematopoietic cells, bone marrow from mice treated with 5-fluorouracil to activate bone marrow precursor cells was harvested and transduced with the VEGFR-2 fusion construct. As a control, the empty MIG vector was used. After sorting, transduced GFP-positive cells were plated in IMDM supplemented with 10% FBS with or without 100 nM AP20187, and cell number was counted at days 5, 7, and 14. We found that cell number decreased rapidly, indicating the necessity of cytokines for the survival of bone marrow cells. However, in marrow cells in which the VEGFR-2 construct was dimerized by the addition of AP20187, we observed a smaller decrease in cell number (Fig. 2A). After two weeks in culture, cell numbers in bone marrow control cultures were 2.5-fold lower than the ones in which VEGFR-2 was dimerized. This effect was not observed in VEGFR-2-transduced cells that did not receive AP20187, indicating that dimerization of VEGFR-2 is required for maintaining hematopoietic cell numbers. We next tested whether dimerization of VEGFR-2 has an additive effect on medium supplemented with hematopoietic cytokines that provide optimal growth conditions (30). Transduced bone marrow cells were cultured in medium containing cytokines that are known to induce hematopoietic cell proliferation (IL-3, IL-6, and SCF), with or without 100 nM AP20187 (Fig. 2B). With these growth conditions, we did not observe any significant change when VEGFR-2 was dimerized in comparison with the control cells, suggesting that VEGFR-2 does not signal a proliferative effect that is synergistic with these hematopoietic cytokines.
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To test whether VEGFR-2 can preserve the viability and activity of hematopoietic progenitors in the absence of hematopoietic cytokines, VEGFR-2 and control cells were cultured in cytokine-free medium for 7 and 14 days with or without the addition of dimerizer, after which cells were plated in methylcellulose medium to assay for hematopoietic progenitors. We found that dimerization of VEGFR-2 maintained hematopoietic progenitor potential in liquid culture. Over a 2-week period in culture, we observed an 8-fold decrease in the number of progenitors in control bone marrow cultures. In contrast, when the FKBP-VEGFR-2 construct was dimerized with AP20187, we observed a 3-fold increase in the maintenance of progenitors over control cultures, consistent with the findings in Fig. 2 (Fig. 3A). Although VEGFR-2 dimerization maintained the hematopoietic progenitor population for a period of 2 weeks in the absence of other cytokines, we did not observe a significant change in the proportion of different hematopoietic progenitors as measured by the CFC assay (Fig. 3B). This result suggests that VEGFR-2 promotes hematopoietic cell survival and/or proliferation but does not affect differentiation of hematopoietic progenitors.
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To confirm that VEGFR-2 can independently maintain the multipotent hematopoietic progenitor population, we utilized the CFU-S12 assay following liquid culture of bone marrow cells for 7 days in cytokine-free medium. Colonies were enumerated in each of the spleens harvested 12 days following injection of bone marrow cells (Fig. 4A). As seen in Fig. 4B, VEGFR-2 dimerization resulted in a 5-fold increase in the proportion of CFU-S12 cells, compared with bone control marrow cultures. These results suggest that VEGFR-2 can maintain the activity and viability of primitive hematopoietic progenitors in the absence of other exogenous cytokines.
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VEGFR-2 Does Not Increase S-phase Entry in Hematopoietic PrecursorsIt is known that, in endothelial cells, VEGF can induce cell proliferation. It has been suggested that this effect is mainly mediated through VEGFR-2 (9). We tested whether dimerization of VEGFR-2 also resulted in bone marrow cell proliferation, which could account in part for the delay in the loss of hematopoietic progenitors that we observed. Bone marrow cells were grown in cytokine-free medium for 2 days, then treated with BrdUrd with or without AP20187 for 2 h. Cytospins of cells were then labeled with an anti-BrdUrd antibody (Fig. 5A). We found that dimerization of VEGFR-2 did not result in a greater proportion of cells which incorporated BrdUrd, indicating that VEGFR-2 signaling alone may not be sufficient to induce proliferation of hematopoietic progenitors (Fig. 5B).
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VEGFR-2 Activation Reduces the Number of Apoptotic Cells in Hematopoietic PrecursorsIt has also been shown that VEGF can induce antiapoptotic signaling through phosphatidylinositol 3'-kinase (PI 3-kinase) in endothelial cells subjected to serum deprivation (31). Since we observed a delay in loss of progenitors when VEGFR-2 is dimerized, we postulated that this effect was caused by an inhibition of apoptosis, since VEGFR-2 dimerization alone did not affect proliferation of hematopoietic progenitors. It has been shown that caspase 3 is present in hematopoietic precursor cells and is activated during apoptosis (32, 33). To test whether VEGFR-2 inhibits hematopoietic cell apoptosis, transduced bone marrow cells were subjected to cytokine deprivation and incubated with or without AP20187 for 14 days. At this point, cytospins were made and stained for the activated form of caspase 3 (Fig. 6A). We found that the proportion of apoptotic cells was 2-fold lower in bone marrow cells in which VEGFR-2 was dimerized compared with bone marrow control cultures (Fig. 6B). Hence, inhibition of apoptosis through VEGFR-2 signaling would explain in part the maintenance of hematopoietic progenitors observed.
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VEGFR-2 Activates the PI 3-Kinase and Erk MAP Kinase PathwaysSince VEGFR-2 dimerization reduces the amount of apoptotic cells, we examined signaling pathways known to be induced by VEGF in endothelial cells. In particular, the PI 3-kinase/Akt and the MAP kinase pathways are both implicated in VEGF signaling and have potential roles in cell survival (31, 34). To determine the kinetics of activation of Akt and Erk1/2 by VEGFR-2, endothelial cells transduced with MIG or MIG-FKBP/VEGFR-2 were starved overnight in medium supplemented with 5% FBS and then treated with AP20187 for 060 min. Membranes were reprobed with total Akt or Erk as a loading control. Following dimerization of VEGFR-2, we found that both Akt and Erk1/2 were activated. Akt phosphorylation peaked between 10 and 20 min (Fig. 7A), whereas maximum Erk1/2 phosphorylation was observed between 20 and 30 min (Fig. 7B). Activation of Akt was biphasic, with a second peak of phosphorylation after 60 min (Fig. 7A). This biphasic activation of Akt in response to VEGFR-2 dimerization was observed in three independent experiments. We next checked whether the Akt and Erk1/2 pathways were also induced in murine bone marrow cells. Transduced GFP-positive bone marrow cells were incubated for 2 days in cytokine-free medium and then stimulated for 20 min with 100 nM AP20187. As with endothelial cells, we also observed activation of Akt (Fig. 7C) and Erk1/2 (Fig. 7D) in bone marrow following VEGFR-2 dimerization. Either of these kinases could account, at least in part, for the survival that we observed in response to VEGFR-2 dimerization, since induction of these signaling pathways by other hematopoietic cytokines, such as SCF and erythropoietin, has been implicated in hematopoietic cell survival (35-37).
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To specifically study the above signaling pathways in mediating hematopoietic progenitor survival, we used specific inhibitors of each signaling pathway. LY294002 is an inhibitor of PI 3-kinase, which activates Akt, whereas U0126 has been shown to block Erk1/2 MAP kinase phosphorylation by specific inhibition of MEK (38). To determine whether VEGFR-2 mediated survival was mediated through Akt and/or Erk1/2, transduced GFP-positive bone marrow cells were incubated with or without 100 nM AP20187 in the presence of the PI 3-kinase inhibitor LY294002 or the MEK inhibitor U0126 at concentrations that blocked each of these kinases (Fig. 7, C and D). Cell number was monitored over a period of 14 days. We found that inhibition of PI 3-kinase blocked the antiapoptotic effect of VEGFR-2 dimerization induced by cytokine deprivation, indicating the essential role of this pathway in VEGFR-2-mediated survival in bone marrow cells (Fig. 8A). Blockade of PI 3-kinase also inhibited the survival of hematopoietic progenitors induced by VEGFR-2 dimerization (Fig. 8B), further demonstrating the critical role of this pathway in survival of hematopoietic progenitors. In contrast, blockade of the MAP kinase pathway with U0126 did not inhibit VEGFR-2-induced cell survival (Fig. 8C). Interestingly, despite minimal effect on cell survival, inhibition of Erk1/2 partially inhibited hematopoietic progenitor activity mediated by VEGFR-2 (Fig. 8D). This discrepancy suggests that hematopoietic progenitors are more dependent on the Erk1/2 MAP kinase pathway for survival than more mature/differentiated cells, which constitute the majority of cells present after 14 days in liquid culture (data not shown).
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DISCUSSION |
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Little is known about the effect of VEGF on adult hematopoiesis, although both VEGFR-1 and VEGFR-2 have been described in hematopoietic precursors (10, 43). Recent reports demonstrate that VEGF, in addition to being a critical regulator of vasculogenesis and hematopoiesis in the embryo, can promote the survival of adult hematopoietic stem cells (14, 44).
Although many studies postulate that VEGF mediates most of its effects through VEGFR-2, it is difficult to make a clear statement about this because of the complexity of the VEGFVEGFR system. To specifically study the effects of VEGFR-2 signaling, we used a strategy that allowed the specific activation of the VEGFR-2 signaling domain by using a chemical inducer of dimerization. This strategy has been successfully used before to specifically study the effects of hematopoietic receptors such as Mpl, Flt-3, granulocyte colony-stimulating factor receptor, and c-Kit on cell proliferation and differentiation (2123). These studies have shown that Mpl can induce long term proliferation of murine hematopoietic progenitors, whereas the effects of Flt-3 and granulocyte colony-stimulating factor receptor are much more modest (23). We therefore used a construct containing the intracellular domain of VEGFR-2, which includes the tyrosine kinase domain, fused to domains that can dimerize in response to the chemical inducer of dimerization, AP20187. This construct was localized to the cytoplasmic membrane in unstimulated HMEC-1 cells and translocated into the cytosol with concomitant phosphorylation following stimulation with AP20187, a phenomenon observed with many endogenous hematopoietic receptors. Interestingly, however, Otto et al. (45) have shown that membrane localization of the thrombopoietin receptor, Mpl, is not required for the full range of Mpl function in hematopoietic cells.
We were interested in the role of VEGFR-2 in hematopoietic progenitors, since little is known about the effects of this receptor on hematopoiesis. In murine bone marrow, our results indicate that VEGFR-2 can maintain hematopoietic progenitor potential following dimerization, since cells fail to survive in the absence of AP20187. It is interesting to note that when hematopoietic progenitors were cultured in the presence of hematopoietic cytokines (IL-3, IL-6, and SCF), there was no effect of VEGFR-2 dimerization on cell number. This would suggest that VEGFR-2 does not induce a proliferative signal in hematopoietic progenitors that is distinct from IL-3, IL-6, and SCF. Although Erk1/2 MAP kinase was activated, VEGFR-2 dimerization failed to induce proliferation of bone marrow progenitors, indicating that signals provided by other factors may be necessary for the proliferation of hematopoietic cells. It is likely then that the delay in cell loss observed through VEGFR-2 dimerization reflects an effect on cell survival. This was verified when we found that VEGFR-2 decreased the fraction of apoptotic cells when hematopoietic progenitors were cultured in the absence of exogenous cytokines.
The importance of cell survival in the maintenance of hematopoietic progenitors was further demonstrated by blocking the PI 3-kinase pathway. This signaling pathway, through Akt, has been shown to play a crucial role in cytokine-mediated survival (31, 46), as well as in the self-renewal of primary multipotential hematopoietic progenitors (47). Blockade of PI 3-kinase completely abolished the maintenance of hematopoietic progenitors mediated by VEGFR-2, indicating that this pathway is critical for VEGFR-2-mediated survival in bone marrow progenitor cells. In contrast, we did not observe a significant effect on cell survival mediated by VEGFR-2 when the Erk1/2 MAP kinase pathway was blocked using the MEK inhibitor, U0126. This would imply that, although the Erk1/2 MAP kinase pathway has been shown to play a role in apoptosis prevention (48), the PI 3-kinase pathway through Akt is the main regulator of VEGFR-2-induced survival signaling in hematopoietic progenitors. However, it is noteworthy that inhibition of Erk1/2 activation did reduce the number of hematopoietic progenitors as measured by the CFC assay. This effect may be explained by the fact that most cells remaining after 14 days in culture in the absence of exogenous cytokines are differentiated, and studies have shown that the cytokine-induced survival of differentiated cells, such as macrophages, is mediated mainly by the PI 3-kinase pathway and not the Erk1/2 MAP kinase pathway (49, 50). Thus, this may indicate that hematopoietic progenitors are more dependent on the Erk1/2 MAP kinase pathway for VEGFR-2-induced survival than more mature cells.
Although VEGFR-2 signaling promoted survival of hematopoietic progenitors and maintained their progenitor potential, it did not seem to affect the differentiation of those progenitors. In contrast, other hematopoietic cytokine receptors, such as Mpl, induce a dramatic expansion of multipotential progenitors and megakaryocytes (21, 23). A recent study has demonstrated that a combination of signals, JAK2 plus either c-Kit or Flt-3 together, can support extensive hematopoietic progenitor cell self-renewal although neither of these receptors can sustain the growth of bone marrow cells alone (47). Whether VEGFR-2 requires additional signals to induce cell proliferation in hematopoietic cells remains unknown, and further studies would be needed to assess this issue.
The distinctions between the roles of the two VEGF receptors in mediating VEGF effects in hematopoiesis still need to be clarified. Both receptors are expressed on subsets of hematopoietic cells such as hematopoietic stem cells, monocytes, and platelets for VEGFR-1 (43, 51) and hematopoietic stem cells, endothelial progenitors, and platelets for VEGFR-2 (10, 51). This overlap in VEGF receptor expression makes it difficult to specifically study the role of each individual receptor. Recent reports have shed some light on the role of VEGFR-1 and its effects on processes such as angiogenesis, hematopoiesis, and inflammation. VEGFR-1 has been shown to promote hematopoiesis by recruiting hematopoietic stem cells from the bone marrow, which favors differentiation and mobilization. Moreover, inhibition of VEGFR1 blocked hematopoietic stem cell cycling, differentiation, and hematopoietic recovery after BM suppression, revealing a function for VEGFR-1 signaling during hematopoiesis (43). VEGFR-2 is implicated in the recruitment and differentiation of endothelial progenitors (11, 52). However, despite its critical role in the development of the hematopoietic system, little is known about the role of VEGFR-2 in adult hematopoiesis. In this paper, we show that VEGFR-2 can induce a protective effect in hematopoietic progenitors. Although VEGFR-2 has been reported to be the main effector of VEGF signaling in endothelial cells, promoting proliferation, survival, and migration, it appears that both VEGF receptors play distinct and important roles in adult hematopoiesisis, and further studies will be needed to clarify this issue.
The strategy used in this study allowed us to demonstrate, for the first time, that VEGFR-2 can activate the PI 3-kinase and Erk1/2 pathways, without any interaction with other VEGF receptors such as the neuropilins or VEGFR-1, in hematopoietic progenitors. Our results show that VEGFR-2 can induce maintenance of hematopoietic progenitors in the absence of exogenous hematopoietic cytokines. This may help to explain, at least in part, the critical role of VEGFR-2 in embryonic hematopoiesis, in which this receptor may promote the survival of early hematopoietic precursors.
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FOOTNOTES |
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¶ Supported by a Doctoral Research Award from the Heart and Stroke Foundation of Canada.
A clinician-scientist of the Canadian Institutes of Health Research and a scholar of the Michael Smith Foundation for Health Research. To whom correspondence should be addressed: Dept. of Medical Biophysics, British Columbia Cancer Research Centre, 601 W. 10th Ave., Vancouver, British Columbia V5Z 1L3, Canada. E-mail: akarsan{at}bccancer.bc.ca.
1 The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; PI 3-kinase, phosphatidylinositol 3'-kinase; MAP, mitogen-activated protein kinase; Erk, extracellular-regulated kinase; Flt, Fms-like tyrosine kinase; FKBP, FK506-binding protein; FBS, fetal bovine serum; EGF, epidermal growth factor; GFP, green fluorescent protein; SCF, stem cell factor; IL, interleukin; IMDM, Iscove's modified Eagle's medium; CFC, colony-forming cell; CFU-S12, colony-forming unit-spleen; BrdUrd, bromodeoxyuridine; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; HA, hemagglutinin; MIG, murine stem cell virus-internal ribosome entry site-enhanced green fluorescent protein.
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ACKNOWLEDGMENTS |
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REFERENCES |
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