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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Vascular Biology, Institute of
Development, Aging and Cancer, Tohoku University, Sendai, Japan; and
the Department of Molecular Genetics, Graduate School of Medicine,
Kyoto University, Japan.
Using polymerase chain reaction-coupled subtractive
hybridization, we have isolated genes expressed during the in vitro
differentiation of murine embryonic stem cells into endothelial cells
(ECs). Among the genes obtained, we identified one gene that was
inducible by vascular endothelial growth factor (VEGF) in the murine EC line MSS31. Analysis of the nucleotide and deduced amino acid sequences
revealed that the protein was composed of 930 amino acids,
including an HEXXH(X)18E consensus sequence of the M1 aminopeptidase family, and is thought to be a mouse orthologue of
puromycin-insensitive leucyl-specific aminopeptidase (mPILSAP). The
recombinant protein hydrolyzed N-terminal leucyl and methionyl residues
from synthetic substrates. Immunohistochemical analysis revealed that
mPILSAP was expressed in ECs during postnatal angiogenesis. Specific
elimination of mPILSAP expression by antisense oligodeoxynucleotide
(AS-ODN) attenuated VEGF-stimulated proliferation, migration, and
network formation of ECs in vitro. Moreover, AS-ODN to mPILSAP
inhibited angiogenesis in vivo. These results suggest a novel function
of mPILSAP, which is expressed in ECs and plays an important role in angiogenesis.
(Blood. 2002;99:3241-3249) The vascular system is the first functional organ
to develop in the embryo. During the formation of the first vascular
system (vasculogenesis), a subset of mesodermal cells, termed
hemangioblasts, aggregate and differentiate into an external layer of
endothelial cells (ECs) and an inner core of blood cells. In the
ensuing process of angiogenesis, new vessels are generated from
the primary capillary plexus by sprouting and intussuception and become
distributed throughout the entire embryo. In the final process of
vascular development, mesenchymal cells surround the blood vessels and differentiate into smooth muscle cells or pericytes, and as a result,
mature, stabilized vessels are established.1 In contrast to embryonic vessels, blood vessels in the adult are composed of ECs
and mural cells and are stabilized. Angiogenesis in the adult commences
with the detachment of pre-existing mural pericytes from pre-existing
microvessels (vascular destabilization), followed by vascular
remodeling, which includes sprouting and intussuception and vascular
stabilization by reattachment of pericytes.2
A number of molecules have been reported to act on ECs and regulate
vascular development in the embryo and angiogenesis postnatally. These
factors include the following: vascular endothelial growth factor (VEGF)3,4 and its receptors fms-like tyrosine
kinase 1 and fetal liver kinase 1 (Flk-1)5,6; angiopoietins and their receptor tyrosine
kinase with immunoglobulinlike loops and epidermal growth factor
homology domain 2;7-9 ephrinB2 and its receptor,
EphB4;10,11 transforming growth factor- In vitro systems of EC differentiation from totipotent embryonic stem
(ES) cells are useful for analyzing vascular development at both the
cellular and the molecular level.17-19 The most popular system is one involving EC-derived embryoid body
formation.17,20 However, the existence of a variety of
cell types other than ECs in embryoid bodies makes it difficult to
analyze, in detail, the cellular and molecular events occurring in ECs
during vascular development. To overcome these difficulties, Nishikawa
et al21 and Hirashima et al22 recently
developed a system that allows ES cells to differentiate into ECs under
2-dimensional culture conditions. Flk-1+/vascular
endothelial (VE)-cadherin To explore the molecular mechanisms of vascular development and
angiogenesis, we isolated genes expressed exclusively during EC
differentiation in this ES cell culture system. Polymerase chain
reaction (PCR)-coupled subtractive hybridization was used to identify
genes abundantly expressed in the
Flk-1+/VE-cadherin+ cell population compared
with Flk-1+/VE-cadherin Materials
The ES cell line CCE was maintained and induced to differentiate into
ECs as described previously.22 Mouse EC line
MSS31,23 a spontaneously immortalized cell line from
spleen ECs, was cultured in Preparation of complementary DNAs from
Flk-1+/VE-cadherin PCR-coupled subtractive complementary DNA cloning With the complementary DNAs (cDNAs) from Flk-1+/VE-cadherin cells as driver and from
Flk-1+/VE-cadherin+ cells as tester,
PCR-coupled cDNA subtraction was performed with a PCR-select cDNA
subtraction kit (Clontech, Palo Alto, CA), used according to the
manufacturer's instructions. The cDNAs abundant in
Flk-1+/VE-cadherin+ cells were inserted into
pCR2.1-TOPO vector by means of a TOPO TA cloning kit
(Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The nucleotide sequences of approximately 250 clones were
determined on both strands by means of a DNA sequencing kit (Applied
Biosystems, Foster City, CA) and an ABI Prism 310 DNA sequencer
(Applied Biosystems) and was analyzed by the BLAST program. With the
cDNA from MSS31 cells as template, full-length cDNA was obtained by
both 5' and 3' rapid amplification of cDNA ends (RACE) with a Marathon
cDNA amplification kit (Clontech), used according to the
manufacturer's instructions. For the amplification of the 5' part of
mPILSAP cDNA, a specific reverse primer
(5'-AAACCCAGTGGGTTATCCATTGGCCTGGAAG-3') and AP1 primer supplied in the
kit were used. For amplification of the 3' region of mPILSAP cDNA, a
specific reverse primer (5'-ACGTGCTCTGGTGGAAAACTGGTCTGTTGTT-3') and AP1
primer were used. PCR products were subcloned, sequenced, and analyzed
as above.
Reverse transcription-PCR analysis We amplified 500 ng cDNA from Flk-1+/VE-cadherin cells or
Flk-1+/VE-cadherin+ cells with an Advantage 2 PCR Enzyme system kit (Clontech) according to the manufacturer's
instructions. The conditions of PCR were as follows: initial
denaturation at 95°C for 5 minutes; 35 cycles of denaturation at
95°C for 1 minute, annealing at 60°C for 1 minute, and extension at
72°C for 1 minute; and a final extension at 72°C for 10 minutes.
The following primers were synthesized and used for amplification: for
mPILSAP, forward primer corresponding to exon 12, 5'-GAT-
GATGGATGGGCTTCTCT-3', and reverse primer corresponding to exon
15, 5'-GGCTTTTCTCAGTACTAGAC-3'; and for mouse
glyceraldehyde-3-phosphate dehydrogenase (G3PDH), forward primer
5'-ACCACAGTCCATGCCATCAC-3' and reverse primer
5'-TCCACCACCCTGTTGCTGTA-3'. PCR products were loaded and
electrophoretically separated on 2% agarose gels containing 0.5 µg/mL ethidium bromide solution.
Northern blot analysis Northern blot analysis was carried out as described previously.24 Briefly, total RNA was extracted by Isogen according to the manufacturer's instructions. Total RNA was separated on a 1% agarose gel containing 2.2 M formaldehyde and transferred to Hybond N+ membrane. The membrane was hybridized with 32P-labeled full-length mPILSAP cDNA probe in hybridization solution at 42°C for 24 hours. After hybridization, the membrane was washed once with 0.1% sodium dodecyl sulfate (SDS) in 2 × SSC at room temperature and then 3 times with 0.1% SDS in 0.2 × SSC at 65°C. Autoradiography was carried out on an imaging plate and analyzed by FLA 2000 (Fuji Film, Tokyo, Japan).Assay for aminopeptidase activity The EcoRI fragment of the mPILSAP cDNA from pCR2.1-TOPO/mPILAP was subcloned into the expression vector pcDNA4/HisMax (Invitrogen). COS-7 cells (4 × 106 cells per 100-mm dish) were transfected according to the manufacturer's instructions with 10 µg pcDNA/mPILSAP mixed with Lipofectin. Transfected COS-7 cells were cultured in 10% FCS/DMEM for 48 hours, washed twice with phosphate-buffered saline (PBS), collected by centrifugation at 500g for 10 minutes, and suspended in PBS. Cells were homogenized by ultrasonication and lysates centrifuged for 15 minutes at 10 000g at 4°C. Supernatant was loaded onto a nickel-chelated resin column, and the histidine-tagged protein was purified by means of an Xpress System Protein Purification Kit (Invitrogen) according to the manufacturer's instructions. We added 1 µg purified protein to 100 µL 25 mM Tris-HCl (pH 7.5), and reactions were initiated by adding an equal volume of Tris-HCl containing 0.5 mM aminoacyl-MCA. After incubation at 37°C for 30 minutes, aminopeptidase activity was measured with a Fluoroskan Ascent (Dai Nippon-Pharmaceuticals, Osaka, Japan) operated at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. For inhibitor assays, the indicated substances were preincubated with the enzyme on ice for 5 minutes, and the reaction was started by adding 0.5 mM leucyl-4-MCA.Subcellular fractionation COS-7 cells transiently transfected with mPILSAP expression vector were suspended in buffer A (30 mM Hepes [pH 7.4], 2 mM EDTA, 5 mM ethyleneglycotetraacetic acid); homogenized in a Dounce-homogenizer; and centrifuged for 10 minutes at 800g. The resulting cell lysates were loaded onto a sucrose cushion (40% sucrose in buffer A) and centrifuged at 100 000g for 60 minutes at 4°C. The supernatant was collected as the cytosolic fraction; the interface, as the plasma membrane (PM) fraction; and the pellet, as the microsomal fraction. The PM fraction was pelleted by centrifugation at 100 000g for 30 minutes at 4°C and solubilized in solubilization buffer (20 mM Tris-HCl, [pH 7.5], 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride). Protein concentration was determined by use of the DC protein assay (Bio-Rad, Hercules, CA). We subjected 40 µg each fraction to Western blot analysis.Western blot analysis Polyclonal rabbit antibody against a synthetic peptide corresponding to the C-terminal 15 amino acids of mPILSAP, including a cysteine linker (CYQSSLSSTEKSQIE), was prepared (anti-mPILSAP Ab). For the detection of mPILSAP in MSS31 cells, cells were starved for 24 hours in 0.1% FCS/ MEM and then stimulated with 100 ng/mL VEGF for 8 hours. The protein extracted from the cells was separated by
SDS-polyacrylamide gel electrophoresis on a 7.5% separating gel and
transferred to nitrocellulose membranes. The membranes were blocked for
1 hour at room temperature with Tris-HCl-buffered saline (TBS), pH
7.4, containing 5% skim milk, and then incubated for 1 hour at room
temperature in TBS containing 0.05% Tween 20 (T-TBS), 1% bovine serum
albumin (BSA), and anti-mPILSAP Ab diluted to 1/2000. The filter was
washed 4 times with T-TBS and incubated for 1 hour with horseradish
peroxidase-conjugated protein G (Bio-Rad) diluted to 1/3000 in T-TBS.
After the filter had been washed 3 times with T-TBS, blots were
detected by an enhanced chemiluminescence method by means of an ECL
Western blotting detection kit (Amersham). The results were visualized
by using the LAS-1000 (Fuji Film).
ODN transfection MSS31 cells were rinsed with Opti-MEM and incubated for 6 to 24 hours with synthetic phosphorothioate ODNs in Lipofectin containing Opti-MEM at a fixed ratio of ODNs versus Lipofectin (1:3; eg, 500 nM oligo per 1500 nM Lipofectin). Thereafter, ODN-containing medium was replaced with normal 0.1% FCS/MEM with or without VEGF.
The sequences of ODNs used in this study were as follows: AS-ODN,
5'-TATGTGGTCAGTCCCCAGTT-3, which is complementary to the messenger RNA
(mRNA) region in mPILSAP; sense (S-ODN), 5'-AACTGGGGACTGACCACATA-3'; mismatched (MIS-ODN), 5'-TTTGTGGACAGTCGCCACTT-3', comprising AS-ODN with 4 base pairs substituted; and scrambled (SCR-ODN),
5'-CTGTATCAGTCAGTGTTCGC-3', comprising all the base pairs of the
antisense codons in random order. AS-ODN was not homologous to any
genes checked against the reported DNA sequences present in the
BLAST program.
Cell proliferation MSS31 cells were inoculated at a density of 2 × 103 per well into 96-well plates and cultured in 10% FCS/MEM for 24 hours. Cells were subsequently treated for 24 hours with or without 500 nM ODN. Following this, the medium was
changed to 0.1% FCS/MEM with or without 100 ng/mL VEGF, and the
cells were incubated for the desired period. We added 10 µL
TetraColor ONE (Seikagaku, Tokyo, Japan) to each well, and
proliferation was quantified by means of a multiple-plate reader
(Tosoh, Tokyo, Japan) set at 492 nm.
Cell migration Cell migration was examined as described previously.25 Briefly, confluent monolayers of MSS31 cells were treated for 6 hours with or without 500 nM ODN, and then wounded with a razor blade. After rinsing with PBS, medium was changed to 0.1% FCS/MEM with or without 30 ng/mL VEGF, and the cells were then
incubated for 24 hours. The number of cells that had migrated across
the wound edge was counted.
Network formation MSS31 cells were cultured in 0.1% FCS/MEM for 24 hours and
were subsequently treated for 6 hours with or without 1000 nM ODNs.
Cells were harvested with 0.25% trypsin and 1 mM EDTA, resuspended in
0.1% FCS/MEM with or without 100 µg/mL VEGF in a final volume of
300 µL, replated (2 × 105 cells per well) onto 24-well
plates coated with Matrigel (250 µL per well), and incubated at
37°C for 4 hours. Cells were observed by phase-contrast microscopy.
The lengths of network structures were quantified with
microcomputer-assisted NIH Image (National Institutes of Health,
Bethesda, MD).
Mouse angiogenesis model A mouse angiogenesis model was prepared as described previously.24 Briefly, male C57BL/6 mice at 4 weeks of age were used. We injected 300 µL growth factor-reduced Matrigel containing VEGF (100 ng/mL) or bFGF (100 ng/mL) plus heparin (32 U/mL) in liquid form at 4°C into the abdominal subcutaneous tissue at the midperitoneal area of each mouse. In some experiments, 50 µM AS-ODN or S-ODN was mixed with the Matrigel. On day 6 after injection, mice were killed and gels recovered. The gels were fixed in 4% paraformaldehyde in PBS and embedded in paraffin. We subjected 3-µm sections of the gels to hematoxylin and eosin staining and immunohistochemical analysis.Immunohistochemical analysis Immunohistochemistry of the sections of the gel was performed as described previously.26 Briefly, anti-mPILSAP Ab, anti-vWF Ab, and nonimmune rabbit immunoglobulin (Ig)-G were used as primary antibody. They were diluted 200-fold with PBS containing 1% BSA. The secondary antibody was biotin-conjugated antirabbit antibody diluted 200-fold with the same diluent. Each reaction was conducted for 30 minutes at room temperature. Coupling of streptavidin to biotin from an ABC kit (Vector Laboratories, Burlingame, CA) was performed for 45 minutes at room temperature. Immunocomplexes were visualized by means of 3,3'-diamidobenzidine tablet sets (Sigma).Quantification of angiogenesis in the Matrigel We measured the hemoglobin contents of the Matrigel from each group; each group contained 4 animals according to the method of Majima et al27 with the following modifications as a parameter for angiogenesis. Briefly, the Matrigels were weighed and homogenized in distilled water (10 µL/mg wet weight). The hemoglobin concentration in the supernatant after centrifugation at 5000g for 5 minutes was determined by a hemoglobin B-test kit (Wako, Osaka, Japan).Calculations and statistical analysis The statistical significance of differences in the data were evaluated by use of unpaired analysis of variance, and P values were calculated by the unpaired Student t test. P < .05 was accepted as statistically significant.
Murine ES cells spontaneously differentiate into ECs in vitro, in
a process that can be monitored by the sequential expression of Flk-1
followed by VE-cadherin.21,22 We defined
Flk-1+/VE-cadherin
We constructed the full-length cDNA of this gene by the RACE method for
sequencing. The nucleotide sequence and the deduced amino acid sequence
are shown in Figure 2. The first ATG
triplet was considered to be the initiation codon, as the
5'-untranslated region contained an in-frame stop codon preceding this
initiation codon. The cDNA contained a 2790-base pair open reading
frame encoding a protein with 930 amino acids. The deduced amino acid sequence contained a consensus sequence typical of the
zinc-aminopeptidase family; this sequence bears an essential
zinc-binding site HEXXH(X)18E of the peptidase at amino acid residues
342 through 346 with a second glutamic acid separated by 18 amino acids
(marked by the box in Figure 2). This motif is found in
aminopeptidase sequences and is a criterion for classification in the
M1 family of aminopeptidases.28 Homology search of the
amino acid sequence revealed that it was 93.4% homologous to
rat29 and 84.1% homologous to human PILSAP,30 respectively (Figure 3). Therefore, this
molecule is proposed as a murine orthologue of PILSAP (mPILSAP).
Next, we examined whether this protein exhibits aminopeptidase
activity. The protein isolated from COS-7 cells transfected with this
gene was measured for relative hydrolytic activity toward various
aminoacyl-MCAs. As shown in Figure 4, the
protein was shown to have aminopeptidase activity. Leucyl-MCA was the
most efficiently hydrolyzed, followed by methionyl-MCA, but little activity was observed toward other aminoacyl-MCAs. To further characterize the enzymatic activity of mPILSAP, we measured
aminopeptidase activity in the presence of various inhibitors
(Table 1). The mPILSAP
was highly sensitive to leucinethiol and zinc ions. General aminopeptidase inhibitors, such as bestatin and amastatin,
significantly inhibited the enzymatic activity of mPILSAP. Puromycin (a
puromycin-sensitive aminopeptidase inhibitor),
1,10-phenanthroline (a chelating agent), leupeptin (a
serine/cysteine protease inhibitor), aprotinin (a serine protease
inhibitor), and pepstatin (an aspartic acid protease inhibitor) had
little effect. Interestingly, fumagillin, an inhibitor of angiogenesis,
inhibited the enzymatic activity of mPILSAP. The analysis of
subcellular localization revealed that mPILSAP was present in cytosolic
and microsomal fractions but not in the membrane fraction (data not
shown). Immunostaining and confocal microscopic observation further
confirmed that mPILSAP was localized to the cytoplasm (data not shown).
By Northern blot analysis using poly(A)+ RNA to examine the expression
of mPILSAP in mouse embryonic and adult tissues, we detected
3.2-kilobyte (kb), 4.0-kb, 4.5-kb, and 5.2-kb transcripts (Figure
5). Expression of mPILSAP in embryonic
development peaked at 7 days after coitus (dpc) and decreased
thereafter (Figure 5A). In the adult, mPILSAP was constitutively and
abundantly expressed in both the liver and the heart. The expression
was also observed in spleen, lung, kidney, and testis, whereas
expression was very faint in skeletal muscle and brain (Figure 5B). As
mPILSAP was inducible in ECs by VEGF, we examined whether mPILSAP was
expressed in ECs during angiogenesis. When Matrigel containing VEGF was inoculated into mouse subcutaneous tissue, new vessels invaded the gel.
Immunohistochemistry with anti-mPILSAP Ab revealed positive staining
of mPILSAP protein in ECs of new vessels, whereas nonimmune rabbit IgG
gave a negative reaction (Figure 6A,C).
ECs of vessels surrounding the Matrigel were faintly stained with
anti-mPILSAP Ab (data not shown). Immunostaining with anti-vWF Ab
confirmed that almost all cells in Matrigel were ECs (Figure 6B).
Next, we constructed ODNs to examine whether mPILSAP was required for
angiogenesis. Northern blot analysis revealed that one AS-ODN
effectively and selectively inhibited mPILSAP expression in MSS31 cells
in response to VEGF stimulation. Corresponding controls of S-ODN,
MIS-ODN, and SCR-ODN did not affect the induced expression of mPILSAP
(Figure 7A). The synthesis of mPILSAP
protein was also inhibited by AS-ODN (Figure 7A). Proliferation and
migration are principal properties of ECs in angiogenesis, and VEGF
augments the degree to which they occur. Therefore, we tested
whether AS-ODN would affect these properties. As shown in Figure 7B,
AS-ODN inhibited the proliferation of MSS31 cells at days 1, 2, and 3, whereas S-ODN, MIS-ODN, and SCR-ODN exhibited no effect. AS-ODN
inhibited the proliferation in a dose-dependent manner over the range
of 1 to 1000 nM (data not shown). Likewise, AS-ODN inhibited the migration of MSS31 cells, whereas S-ODN, MIS-ODN, and SCR-ODN had no
effect (Figure 7C). Again, AS-ODN showed a dose-dependent inhibition of
cell migration, over the range of 1 to 1000 nM (data not shown). We
further examined the effect of AS-ODN on angiogenesis. We used network
formation of MSS31 cells as an in vitro model of angiogenesis. AS-ODN
inhibited the network formation of MSS31 cells, whereas S-ODN, MIS-ODN,
and SCR-ODN had no effect (Figure 8A),
and this was further confirmed by quantitative analysis (Figure 8B).
Finally, we used a mouse model of angiogenesis to confirm the
requirement of mPILSAP in angiogenesis in vivo. The Matrigel plug assay
is widely accepted as an in vivo model of angiogenesis, and the direct
application of antisense phosphothioate oligonucleotides is easily
achieved in this assay.31 Matrigel containing VEGF and
either AS or S-ODN was inoculated into mouse subcutaneous tissue.
Invasion of new vessels into the gel was enhanced by VEGF (Figure
9A; panel 9Ai versus 9Aii).
AS-ODN but not S-ODN inhibited this process (Figure 9Aiii,9Aiv).
Similar results were obtained when bFGF was used as an angiogenic
factor (Figure 9Av-9Aviii). Quantitative analysis confirmed that AS-ODN
significantly inhibited VEGF- and bFGF-stimulated angiogenesis in vivo,
whereas S-ODN exhibited no inhibition (Figure 9Bi,9Bii).
We found that mPILSAP expression was augmented during in vitro differentiation of ECs from murine ES cells. VEGF induced mPILSAP in ECs in vitro, and the expression of mPILSAP was confirmed in ECs during postnatal angiogenesis in vivo. Moreover, the specific elimination of mPILSAP expression by AS-ODN abrogated proliferation and migration of ECs in vitro, as well as angiogenesis in vivo. These results indicate that mPILSAP plays an important role in angiogenesis. Although bFGF did not significantly induce mPILSAP in ECs in vitro, the angiogenic activity of bFGF in vivo was also significantly inhibited by AS-ODN to mPILSAP. The reason for this inhibition is not clear at the moment. The angiogenic activity of bFGF in vivo is reported to be dependent on the endogenous VEGF.31 Thus, it is possible that AS-ODN to mPILSAP inhibited bFGF-stimulated angiogenesis by inhibiting the effect of endogenous VEGF. Aminopeptidases catalyze the sequential removal of amino acids from unblocked N-termini of peptides and proteins and play important roles in various biological processes, such as maturation, activation, modulation, and degradation of bioactive peptides, and in the determination of protein stability.32 The mPILSAP belongs to the M1 family of aminopeptidases, which contain an HEXXH(X)18E motif and a central Zn2+ ion essential for their enzymatic activity. Eight aminopeptidases of this subfamily have been identified to date and are subdivided into membrane-bound and cytoplasmic enzymes. Membrane-bound ectoenzymes include aminopeptidase N (APN)/CD13,33 aminopeptidase A,34 thyrotropin-releasing-hormone-degrading ectoenzyme,35 and insulin-regulated membrane aminopeptidase/human placental leucine aminopeptidase/oxytocinase.36 Cytoplasmic enzymes include aminopeptidase B,37 leukotriene-A4 hydrolase,38 puromycin-sensitive aminopeptidase,39 and PILSAP.29,30 We confirmed that mPILSAP was present in cytoplasmic and microsome fractions but not in the membrane fraction when expressed in COS-7 cells. The mPILSAP is not the only member of the M1 aminopeptidase thought to play a role in angiogenesis. APN/CD13 is expressed in ECs of tumor vessels. Anti-APN antibodies as well as the aminopeptidase inhibitor bestatin inhibit angiogenesis in vivo.40 APN/CD13 belongs to the membrane-bound ectoenzyme group of aminopeptidases, whereas mPILSAP is present in the cytoplasm. Therefore, APN/CD13 and mPILSAP are thought to play distinct functions. However, the mechanism by which aminopeptidases regulate angiogenesis is currently unknown. Methionine aminopeptidase 1 (MetAP1) and MetAP2 belong to another subset of aminopeptidases, which contain a central Co2+ ion for their enzymatic activity and do not belong to the M1 family of aminopeptidases.41,42 Fumagillin and its derivative TNP-470 are potent inhibitors of EC proliferation and angiogenesis.43,44 The target molecule of fumagillin and of its derivative TNP-470 was recently shown to be MetAP2.45 However, the expression of MetAP2 is not restricted to ECs, and selective inhibition of EC proliferation by fumagillin is not related to differential expression of MetAP2.46 Therefore, it is not clear whether fumagillin and TNP-470 exhibit antiangiogenic activity via the specific inhibition of MetAP2. We observed that fumagillin inhibited the enzymatic activity of mPILSAP. Therefore, it is possible that fumagillin and TNP-470 exert their antiangiogenic activity by inhibiting other aminopeptidases including mPILSAP in ECs. Further study is required to explore whether fumagillin and TNP-470 can bind to mPILSAP and other aminopeptidases besides MetAP2. As mPILSAP was isolated during EC differentiation from ES cells in vitro, we assume that mPILSAP is involved not only in postnatal angiogenesis but also in embryonic vascular development. The expression of mPILSAP in the mouse embryo peaked at 7 dpc, and declined thereafter (Figure 6A). The high expression of VEGF in the mouse embryo is associated with vasculogenesis, whereas its low expression is associated with angiogenesis.47 Thus, mPILSAP expression may correlate with VEGF expression. In contrast, mPILSAP is constitutively and highly expressed in limited organs, such as heart and liver in the adult (Figure 6B). These findings also suggest that the biological function of mPILSAP is not restricted to the vascular system. Further studies, including the targeted disruption and overexpression of the mPILSAP gene in mice, are currently underway to clarify the biological significance of mPILSAP in embryonic vascular development as well as in other organs.
We thank Dr Stuart T. Fraser for critical reading of this manuscript. We are indebted to Ms Hiroko Oikawa for her excellent technical assistance.
Submitted May 29, 2001; accepted December 26, 2001.
Supported by the Japan Society of the Promotion of Science Research for the Future (99L01304) and by the Japanese Ministry of Education, Science, Sports, and Culture.
The nucleotide sequence reported in this paper has been submitted to the DDBJ/EMBL/GenBank with accession number AB047552.
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: Yasufumi Sato, Dept of Vascular Biology, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan; e-mail: y-sato{at}idac.tohoku.ac.jp.
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Abstract
Introduction
and its receptors
