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1 Department of Vascular Biology, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Japan
2 Department of Nanomedicine (DNP), Tokyo Medical and Dental University Graduate School, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan
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Abstract |
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Introduction |
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With use of a subtraction strategy, we previously isolated a mouse version of puromycin insensitive leucyl-specific aminopeptidase (PILSAP), the expression of which is augmented in mouse embryonic stem (ES) cells during in vitro differentiation to mature ECs. Moreover, PILSAP is expressed in ECs at the site of postnatal angiogenesis, and specific elimination of PILSAP expression abrogates proliferation and migration of vascular endothelial growth factor (VEGF)-induced ECs in vitro, as well as VEGF- or basic fibroblast growth factor (bFGF)-induced angiogenesis in vivo (Miyashita et al. 2002). In previous studies, we have demonstrated that PILSAP is required for endothelial integrin function during cell adhesion and migration (Akada et al. 2002) and that PILSAP plays a crucial role in EC proliferation via binding and modification of phosphatidylinositol-dependent kinase 1 (PDK1) (Yamazaki et al. 2004). Experiments involving the introduction of a mutant PILSAP cDNA (mtPILSAP) into ECs revealed that the effects of PILSAP on angiogenesis are dependent on its aminopeptidase activity (Yamazaki et al. 2004). Furthermore we have found that the expression of PILSAP in ECs is regulated, at least in part, by a transcription factor called polyomavirus enhancer-binding protein 2 (PEBP2) (Niizeki et al. 2004).
Aminopeptidases are metalloproteinases that remove amino acids from N-termini of proteins and function during post-translational modification. It has been reported that some aminopeptidases play an important role in angiogenesis (for review see Sato 2004). These enzymes include methionine aminopeptidase type 2 (MetAP2) (Griffith et al. 1997), aminopeptidase N (APN)/CD13 (Bhagwat et al. 2001), aminopeptidase A (APA) (Marchio et al. 2004) and PILSAP/adipocyte-derived leucine aminopeptidase (A-LAP) (Akada et al. 2002; Miyashita et al. 2002; Yamazaki et al. 2004). Three aminopeptidases, APA, APN and PILSAP/A-LAP, belong to the M1 subfamily of aminopeptidases, which contain a consensus HEXXH(18X)E motif for Zn2+ binding. APA and APN are membrane-bound M1 zinc-aminopeptidases. Although APA-null mice fail to respond to hypoxia and angiogenic growth factors, they develop normally (Marchio et al. 2004). To date, there have been no reports demonstrating that any of aminopeptidases play a role in endothelial differentiation.
PILSAP/A-LAP is a cytoplasmic enzyme that differs with respect to its inhibition (puromycin-insensitive) and its unusual substrate specificity (leucine, and to a lesser extent methionine) from the other M1 members (Miyashita et al. 2002). Since PILSAP was isolated as a molecule that was up-regulated during endothelial cell differentiation (Miyashita et al. 2002), we hypothesized that PILSAP pertains to that. In order to explore this, the current research examined the role of PILSAP in endothelial differentiation and vascular morphogenesis using an in vitro three-dimensional ES differentiation system involving embryoid bodies (EBs). Our results suggest that PILSAP increases the growth in population of vascular, hematopoietic and muscle lineages from Fms-like tyrosine kinase-1 (Flk-1)-positive mesodermal precursors and plays a role in vascular morphogenesis.
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Results |
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We cultured ES cells to form EBs, and observed the vascular structure formation and concurrent expression of various biomarkers. The vascular structure was detected by immunostaining with anti-mouse CD31 moAb. The clumps and short strings of CD31-positive cells appeared around day 4 and gradually formed networks to reach a plateau level at day 9–11 (Fig. 1A). Transcription factor Oct3/4, a hallmark of undifferentiated ES cells, was decreased in the initial EB formation (Fig. 1B). The T-Box transcription factor brachyury, a marker of pan-mesoderm (Wilkinson et al. 1990), was expressed from day 3, reached its maximum level at day 4 and thereafter was hardly detectable. As ES cells differentiated, mesodermal induction was further conducted, followed by emergence of an Flk-1-positive mesodermal population (Fig. 1B). Flk-1 is the earliest marker for endothelial cell precursors and is indispensable for vasculogenesis (Yamaguchi et al. 1993). Here we observed that Flk-1 showed two peaks, at day 4 and day 10. The first peak of Flk-1 preceded the peaks of any other EC markers such as CD31, VE-cadherin, CD34, and Tie-2 (Fig. 1B). Expression of CD31 and Tie2 peaked at day 10, whereas the peak days of VE-cadherin and CD34 were both found on day 8. These data suggest that differentiation of ECs terminates around day 10 in our system (Fig. 1B).
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PILSAP was highly expressed at the beginning of the observed period (day 3), decreased thereafter, and increased again through day 10 (Fig. 1B). PILSAP was originally cloned as a molecule which over-expressed in mature ECs (Flk-1+/VE-cadherin+) compared to endothelial progenitor cells (EPCs) (Flk-1+/VE-cadherin–) derived from ES cells in a two-dimensional culture system (Miyashita et al. 2002). This discrepancy might be caused by difference in ES differentiation system. Because PILSAP expression was followed by Flk-1 during the course of ES differentiation (Fig. 1B), we examined the role of PILSAP in vascular morphogenesis and endothelial differentiation.
To examine the effect of PILSAP on vascular morphogenesis, we transfected mutant PILSAP (mtPILSAP) cDNA lacking aminopeptidase activity to ES cells. Mock-ES cells or mtPILSAP-ES cells were cultured in methylcellulose for the endothelial induction as described in Experimental procedures. The vascular structure in EBs made of each transfectant is shown in Fig. 2A. There was no significant difference in size between mock- and mtPILSAP-EBs (data not shown). mtPILSAP-EBs was organized with less vascular structure (Fig. 2B). Moreover, vascular structure in mock-EBs showed a hierarchical pattern of broad vessels tapering to narrow vessels, whereas that in mtPILSAP-EBs showed homogeneous and fine networks (Fig. 2A). To confirm the effect of aminopeptidase activity of PILSAP on vascular development in EBs, we used leucinethiol (LT), which has been shown to inhibit PILSAP aminopeptidase activity (Miyashita et al. 2002), and small interfering RNA (siRNA) for PILSAP. When the enzymatic activity or expression of PILSAP was inhibited by LT or siRNA, vascular network formation was hampered (Fig. 2C–E), although treatment of LT or siRNA did not reduce the size of EBs (data not shown).
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Because expression of PILSAP preceded the first peak of Flk-1 expression (Fig. 4A), we investigated whether PILSAP was required for the initial expression of Flk-1. The expression level of Flk-1 in mtPILSAP-EBs was equivalent to that in mock-EBs on day 4 (the first peak day of Flk-1), whereas Flk-1 was expressed in lower levels in mtPILSAP-EBs than in mock-EBs on day 10 (the second peak day), as shown in Fig. 4B. The degree of inhibition varied among experiments, but the results were the same for all genes evaluated in this study.
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PILSAP is required for the growth in population of other vascular cells than endothelial cells
Alpha smooth muscle actin (
SMA) is a marker of vascular smooth muscle cells (VSMCs), whereas Pim-1 has been reported to be a target of Flk-1 in ES cells and to regulate differentiation of VSMCs (Zippo et al. 2004). These findings are consistent with our result that the expression of Pim-1 followed the appearance of Flk-1 and preceded SMA induction (Fig. 5A). It has been demonstrated that Flk-1 positive cells derived from ES cells differentiate into both ECs and VSMCs (Yamashita et al. 2000). We observed that expression of SMA was preceded by that of Pim-1 (Fig. 5A) and both expressions were suppressed together with that of Flk-1 in mtPILSAP-EBs (Fig. 5C). The expression of SMA in cells of mtPILSAP-EBs was also lower than in Mock-EBs (Fig. 5D). However, juxta-existence of CD31-positive cells and SMA-positive cells was rarely observed even in Mock-EBs.
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It is reported that the in vitro ES culture system in methylcellulose with VEGF generates blast cell colonies (Choi et al. 1998). The cells of these colonies, namely blast colony-forming cells (BL-CFCs), represent the in vitro equivalent of a transient common precursor of hematopoietic and endothelial lineages, the hemangioblast (Choi et al. 1998). We analyzed sequential expression of Flk-1 and hematopoietic lineage markers such as stem cell leukemia (SCL), runt-related gene 1 (Runx1), and CD45 (Fig. 6A).
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Expression of CD45, a pan blood cell marker, was first identified at day 8 and increased up to day 12 (Fig. 6A). The amount of CD45 mRNA in mtPILSAP-EBs was smaller than that in mock-EBs (Fig. 6D). Fig. 6B–D suggests that PILSAP functions in hemangioblast differentiation.
Role of PILSAP in the generation of other germ layer-derived cells
Motoike et al. (2003) reported that Flk-1 positive mesodermal cells can differentiate to striated muscle. The amount of myogenin mRNA in mtPILSAP-EBs was significantly smaller than that in mock-EBs (Fig. 7A). To further investigate the role of PILSAP in differentiation of cells derived from other germ layers, we measured and compared the amount of ectodermal specific mRNA in EBs made of each transfectant. Glial fibrillary acidic protein (GFAP) is a marker of astroglial cells and of neuron progenitors (Garcia et al. 2004), which are derived from ectoderm. The mtPILSAP-EBs showed a level of GFAP expression similar to that for mock-EBs (Fig. 7B).
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Discussion |
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Flk-1 is a marker of mesodermal precursors and ECs. Here we showed for the first time that Flk-1 expression exhibited two peaks, on days 4 and 10, with the first peak preceding the appearance of any of the other EC markers (Fig. 1B). A transient reduction in expression of Flk-1 was shown in a previous report where EBs were harvested at either 24-, 12- or 6-h intervals over a 6-day period using a similar in vitro ES differentiation system (Robertson et al. 2000). The reason why two peaks of Flk-1 expression were not recognized might be because EBs were not adequately analyzed (Lacaud et al. 2002). Hemangioblasts are shown to be a subpopulation of brachyury+ and Flk-1+ cells in mouse embryo (Huber et al. 2004). Thus hemangioblasts are supposed to emerge primarily at day 4 in our in vitro ES differentiation system. Peak expression of EC markers such as CD31, VE-cadherin and Tie-2 appeared by day 10, suggesting that endothelial differentiation terminates by day 10 in our system.
The expression of PILSAP was high in both undifferentiated ES cells (0.79 ± 0.06) and EBs on day 3 (1.00 ± 0.02). It decreased thereafter, but increased again through day 10 (Fig. 1B). In contrast, Oct3/4 simply decreased from the beginning of differentiation and became almost undetectable on day 6. The mRNA of Oct3/4 on day 3 of the peak expression was not altered in mtPILSAP (104 ± 10% compared to 104 ± 3% in Mock-EBs, n = 3). The function of PILSAP in the undifferentiated state remains to be elucidated. Bracyury, a pan-mesodermal marker, was expressed in undifferentiated ES cells (day 0) as little as on day 6 (0.091 ± 0.006 and 0.096 ± 0.004, respectively; 1.00 ± 0.07 was the maximum expression on day 4; n = 3). Although PILSAP expression preceded the expression of brachyury, its expression was not altered in mtPILSAP-EBs at its expression peak, day 4 (118 ± 6% to 100 ± 3% in Mock-EBs, n = 3). Therefore, PILSAP may not be involved in whole mesodermal expression. Because PILSAP was expressed earlier than the first peak of Flk-1 (day 4), we investigated whether PILSAP was involved in initial appearance of Flk-1. The first peak of Flk-1 in mtPILSAP-EBs was not different from that seen with mock-EBs, but the second peak was significantly lower in mtPILSAP-EBs than in mock-EBs (Fig. 4B). Thus, PILSAP is not involved in the first peak of Flk-1 in mesodermal precursors, but it is involved in the subsequent differentiation to ECs (Fig. 3), VSMCs (Fig. 5C), blood cells (Fig. 6) and striated muscle cells (Fig. 7A). It has been reported that cooperative interaction of EPAS1 and Ets-1 is required for full transcriptional activation of Flk-1 in ECs (Elvert et al. 2003), whereas HoxB5 transactivates the Flk-1 promoter and is sufficient to regulate mesodermal differentiation to endothelial lineage (Wu et al. 2003). Expression levels for Ets-1 and EPAS-1 in mtPILSAP-EBs were not restrained on day 4 (the first peak day of Flk-1), but significantly lower on day 10 compared to mock-EBs (Fig. 4D,E). The reduction in expression of Flk-1, EPAS-1 and Ets-1 in mtPILSAP-EBs on day 10 is thought to result from reduction in the number of mature ECs.
VE-cadherin, which was previously thought to be a definitive marker of ECs, is now recognized to be expressed in intermediate hemogenic ECs and maintained in mature ECs, but lost in blood cells (Fraser et al. 2003; Wang et al. 2004). We observed that around day 6 the expression of VE-cadherin was high, whereas other EC markers such as Tie-2 and CD31 showed lower expression (Fig. 1B). The hemogenic ECs may exist in this period. Runx1 was involved in the generation of hematopoietic cells from endothelial cells (Hirai et al. 2005). Furthermore, Hirai et al. (2005) used a proximal promoter of Runx1 to monitor its endogenous expression and found that Runx1 appeared approximately one day later than Flk-1 and repressed Flk-1 expression during transition of hemogenic ECs to hematopoietic cells in an early stage of in vitro ES differentiation system. The transient reduction of Flk-1 expression in our system might be due to the appearance of p-Runx1 (Fig. 4A). A transfection of mtPILSAP into ES cells restrained VE-cadherin expression on day 10, but not on day 6–8 (Fig. 5B), which suggests PILSAP involvement is in the later stage of endothelial differentiation rather than the intermediate stage.
Flk-1 positive cells derived from ES cells are regarded as vascular progenitor cells, because they differentiate into both ECs (CD31+/SMA–) and VSMCs (CD31–/SMA+) (Yamashita et al. 2000). Pim-1 has been identified as a downstream target of Flk-1 by comparing the transcripts differentially expressed in wild-type vs. Flk-1 null ES cells (Zippo et al. 2004). Furthermore, Pim-1 is required for differentiation of ES cells into ECs and mural cells, as well as for VSMC proliferation (Zippo et al. 2004). The expressions of Pim-1 and SMA in mtPILSAP-EBs of day 10 were significantly lower compared with those in Mock-EBs (Fig. 5C) and the number of SMA-positive cells was smaller in mtPILSAP-EBs than in Mock-EBs (Fig. 5D).
Moreover, the expressions of SCL, Runx1 and CD45 were inhibited in mtPILSAP-EBs of day 10. Thus PILSAP is required for differentiation of Flk-1 positive mesodermal precursors to vascular cells (ECs and VSMCs) and hematopoietic cells.
Recently it has been shown that skeletal muscle is also derived from Flk-1 positive mesodermal cells using in vitro differentiation and in vivo transplantation systems (Motoike et al. 2003). We obtained the results consistent with findings given in that report (Fig. 7A). These results indicate that PILSAP regulates differentiation of Flk-1 positive mesodermal progenitor cells to a wide spectrum of lineages, not only vascular and hematopoietic cells, but also skeletal muscle cells. However, expression of GFAP, a neuroglial marker was unchanged in mtPILSAP-EBs compared with mock-EBs (Fig. 7B). Thus, PILSAP does not affect differentiation of cells of ectodermal lineage other than cells of mesodermal lineage.
In summary, our study indicates that PILSAP plays a critical role in the differentiation of Flk-1 positive mesodermal precursors. To best of our knowledge, this is the first indication that an aminopeptidase participates in the differentiation of certain cell lineages. The mechanism how PILSAP functions in differentiation and morphogenesis of the vascular system remains to be elucidated.
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Experimental procedures |
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MG1.19 cell (Gassmann et al. 1995), a CCE ES cell line, was maintained in a culture dish coated with gelatin using Glasgow minimum essential medium (GMEM) (Sigma) supplemented with 10% fetal calf serum (FCS), 1% nonamino acid solution, 1 mM sodium pyruvate, 2 mM L-glutamine, 2000 U/mL leukemia inhibitory factor (LIF), 100 µg/mL G418, and 0.1 mM 2-mercaptoethanol (2-ME).
Supertransfection of PILSAP cDNA into murine ES cell line
We made a mtPILSAP cDNA with a dominant negative activity (Yamazaki et al. 2004) and introduced into pHPCAG (Niwa et al. 1998) to make a construct lacking aminopeptidase activity (pHPCAG-mtPILSAP). MG1.19 cells were transfected with pHPCAG-mock, or pHPCAG-mutant PILSAP and cultured with 50 µg/mL hygromycin B to obtain the permanent transfectants, namely Mock-ES cells and mtPILSAP-ES cells.
Three-dimensional in vitro ES differentiation assay
In vitro differentiation of MG1.19 cells or transfectants in methylcellulose was performed essentially as described elsewhere (Vittet et al. 1997). Briefly, cells were cultured in IMDM Glutamax medium supplemented with 1% methylcellulose, 15% FCS, 450 µM monothioglycerol, 10 µg/mL insulin, 50 units/mL penicillin and 50 µg/mL streptomycin, 50 ng/mL VEGF, 2 units/mL erythropoietin, 100 ng/mL bFGF, and 10 ng/mL interleukin-6. To add reagent repeatedly, we performed another three-dimensional in vitro ES differentiation assay, namely suspension culture. MG1.19 cells were plated into a 96-well low cell binding plate (Nalge Nunc) with differentiation medium (DM) which was MEM supplemented with 10% FCS, 1 nM VEGF, and 5 x 105 M 2-ME in the absence of LIF. The DM containing 10 µM LT or dimethylsufoxide (DMSO) as vehicle was changed on day 0, 2, 5 and 8. In some experiments, cells were transfected with 200 nM siRNA for PILSAP (Serwold et al. 2002) or scramble RNA (scrRNA) as control and incubated for two days. Thereafter, cells were plated into a 96-well low cell binding plate with DM and transfection was conducted on day 3, 6, and 9. EBs were collected on day 12 for whole-mount immunohistochemistry and on day 10 (mtPILSAP and siRNA) and day 12 (LT) for quantitative reverse transcriptase (RT)-PCR analysis.
Whole-mount immunohistochemistry
Whole-mount immunohistochemistry was performed as described elsewhere (Davis 1993) with some modification. CD31-positive vascular structures were visualized using biotinylated anti-mouse CD31 monoclonal antibody (moAb) (eBioscience), streptavidin-horse radish peroxidase, and then 3,3'-diaminobenzidine (DAB) and observed by stereomicroscope (Leica Microsystems). Biotinylated rat IgG2a was used as a negative control. The quantitative analysis of vascular area in EBs was performed using a NIH image software. Vascular length of EBs in suspension culture was measured by analySIS (Soft Imaging System GmbH). In some experiments, EBs were incubated with allophycocyanin (APC)-conjugated anti-mouse CD31 moAb (MEC13.3) and FITC-conjugated anti- SMA moAb (clone 1A4) (Sigma) and observed by a confocal microscopy (LSM510META) (Carl Zeiss).
Real time RT-PCR analysis
First-strand cDNAs were generated from total RNA with 1st Strand cDNA Synthesis Kit for RT-PCR (Roche). Real time RT-PCR was conducted by LightCycler FastStart DNA Master SYBR Green I and LightCycler System (Roche). The primer sets used for amplification are presented as Supplementary Table S1. Each mRNA level was measured as a fluorescent signal intensity and corrected with use of the intensity for ß-actin.
FACS analysis
The EBs were harvested and dissociated by incubating with cell dissociation buffer (Life Technologies) and vigorous pipetting. The harvested cells were incubated in mouse serum for 30 min on ice to block the nonspecific antibody binding, and incubated with R-phycoerythrin (R-PE)-conjugated anti-mouse Flk-1/VEGFR-2 Ab (Avas121) for 15 min on ice. After each step, cells were washed with Hanks’ balanced salt solution (Life Technologies) containing 1% BSA and 0.01% sodium azide (Wako). Living cells excluding propidium iodide (Sigma) were analyzed by FACS Vantage (Becton Dickinson).
Calculations and statistical analysis
The statistical significance of differences in the data was evaluated by use of analysis of variance (ANOVA), with P-values calculated by Tukey's methods. A value of P < 0.05 was accepted as statistically significant.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: mayudnp{at}tmd.ac.jp
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References |
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Received: 29 November 2005
Accepted: 28 March 2006
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