1 Department of Oral and Maxillofacial Surgery, Nagoya Daini Red Cross Hospital,
2-9 Myoken-cho, Showa-ku, Nagoya 466-8650, Japan
2 Department of Biochemistry, Nagoya University School of Medicine, 65
Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
3 Department of Tissue Engineering Nagoya University School of Medicine, 65
Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
4 Department of Oral and Maxillofacial Surgery, Nagoya University School of
Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
* Author for correspondence (e-mail: tmurama{at}med.nagoya-u.ac.jp )
Accepted 26 April 2002
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Summary |
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Key words: Midkine, Collagen gel, Endothelial cell, Interleukin-8, Smooth muscle cell
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Introduction |
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Angiogenesis is one example of epithelial-mesencymal interactions, in which
endothelial cells and smooth muscle cells interact with each other. L'Heureux
et al. reported that they made an artificial blood vessel equivalent
constructed with human vascular smooth muscle cells, endothelial cells and
fibroblasts (L'Heureux et al.,
1993). Their tissue-like structure was obtained by the contraction
of a tubular collagen gel by vascular smooth muscle cells, generating a
media-like structure, and endothelium was established within the tubular
structure after intraluminal cell seeding. We modified their artificial blood
vessel equivalent, and developed an artificial blood vessel model (BVM), which
is described in this paper. Our BVM was made up of collagen gel populated with
human aortic smooth muscle cells (HASMC) and human umbilical vein endothelial
cells (HUVEC), seeded on the gel. This model enables simple construction of a
blood vessel-like structure, and may be used to examine the activities of
exogenously added factors in tissue interactions.
Here, we utilized the BVM to analyze the role of midkine (MK), a
heparin-binding growth factor (Kadomatsu
et al., 1988; Tomomura et al.,
1990
), in interactions between HASMC and HUVEC. MK promotes
angiogenesis (Choudhuri et al.,
1997
), neurite outgrowth
(Muramatsu et al., 1993
),
survival of neurons (Owada et al.,
1999
), cell growth (Muramatsu
and Muramatsu, 1991
), fibrinolysis
(Kojima et al., 1995
) and cell
migration (Takada et al.,
1997
; Maeda et al.,
1999
; Horiba et al.,
2000
). MK has been suggested to have roles in
epithelial-mesenchymal interactions in two systems. First, anti-MK antibody
inhibited development of tooth germ in vitro
(Mitsiadis et al., 1995a
).
Second, during branching morphogenesis of embryonic lung in vitro, MK added to
the medium enhanced mesenchymal development (Toriyama et al., 1996). However,
whether MK is involved in inter-tissue interactions remains to be
clarified.
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Materials and Methods |
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Culture of HASMC and HUVEC
HASMC and HUVEC were purchased from Kurabo (Tokyo, Japan). HASMC were
maintained in HuMedia-SG (Kurabo) supplemented with 5% fetal calf serum (FCS;
Hyclone, Logan, UT), 1 ng/ml amphotericin B, 1 ng/ml gentamycin, 1 ng/ml human
recombinant epidermal growth factor (EGF), 2 ng/ml bFGF and 5 µg insulin.
HUVEC were maintained in HuMedia-KG (Kurabo), supplemented with 2.5% FCS, 1
ng/ml amphotericin B, 1 ng/ml gentamycin, 10 ng/ml EGF, 5 µg/ml bFGF, 1
ng/ml hydrocortisone and 10 µg/ml heparin. They were grown at 37°C in
an atmosphere of 5% CO2/95% air. Cells were used for experiments
between the third and fourth passage.
Preparation of BVM
Collagen gels containing HASMC were prepared according to the procedure
described previously (Sumi et al.,
2000). Briefly, medium with 0.2% collagen was prepared by mixing
0.3% pepsin-processed type I atelocollagen solution (Koken Co. Ltd., Tokyo,
Japan), six-fold concentrated minimal essential medium (MEM) (Gibco BRL Life
Technologies Inc., Rockville, MD) and FCS at a ratio of 4:1:1 (V/V/V). HASMC
were dispersed with 0.05% trypsin and 0.02% EDTA (Gibco) in Dulbecco's
phosphate buffered saline (PBS). Cells were suspended at a density of
1.0x105 cells/ml in the medium with 0.2% collagen solution
and dispensed at 3.0 ml/dish into six-well tissue culture plates (Falcon,
Becton Dickinson Labware, Franklin Lakes, NJ). After incubation at 37°C in
5% CO2/95% air for 2 hours, cultures were carefully washed twice
with Dulbecco's modified Eagle's medium (DMEM) (Gibco) and then with 1 ml of
DMEM supplemented with 10% FCS.
HUVEC were dispersed with 0.05% trypsin and 0.02% EDTA in PBS(-), and the cell density was adjusted to 1.0x106 cells/ml with DMEM supplemented with 5% FCS (DMEM-FCS), then seeded on HASMC gels, which were cultured for 7 days with DMEM-FCS at 1 ml/gel (1.0x106 cells). This complex was used as the BVM and was cultured at the air-liquid interface. The BVM was cultured with DMEM-FCS supplemented with MK (0, 10, 50, 100 and 200 ng/ml). The model was also cultured in DMEM supplemented with MK (100 ng/ml) or IL-8 (100, 500, 1000 ng/ml).
Cell proliferation assay
HUVEC and HASMC were maintained in the respective specific medium
containing no supplemental factors except FCS. HASMC were seeded onto 96-well
plates at a density of 0.5x104 cells per well, and HUVEC were
seeded onto 96-well plates at a density of 1.0x104 cells per
well.
To examine whether HASMC-derived soluble factors affect HUVEC proliferation, we employed a co-culture system with polycarbonate membrane (pore size, 0.4 µm) (Transwell 24-well culture plate, Corning Inc. NY). HASMC were seeded onto culture plates at a density of 5.0x104 cells per well and cultured for 24 hours with DMEM supplemented with 5% FCS. Then, HUVEC were seeded onto culture inserts at a density of 1.0x105 cells per well and cultured with DMEM supplemented with 5% FCS.
Cell numbers were assessed using a WST-1 cell counting kit (Wako Chemical Co., Tokyo, Japan), which is based on conversion of the tetrazolium compound to the formazan product by the cells, and numbers were determined at the indicated time points.
Preparation of HASMC-conditioned medium
Confluent HASMC in 10 cm dishes were incubated with 6 ml of DMEM for 48
hours at 37°C. The HASMC-conditioned medium was collected, centrifuged to
remove dead cells and stored at -80°C.
Morphological observations
BVMs were cultured for 24 and 72 hours in DMEM supplemented with 5% FCS
with or without MK (100 ng/ml). Then they were cultured for 24, 48 or 72 hours
in serum-free DMEM with MK (0, 100 ng/ml) or IL-8 (0, 100, 500, 1,000 ng/ml)
and fixed with 4% paraformaldehyde in PBS, pH 7.4. The BVMs were embedded in
paraffin and cut into sections 5 µm thick vertically. Serial sections were
mounted on slides, dried overnight and stored in an airtight box. Sections
were stained with hematoxylin-eosin (H-E) or with a kit to detect
proliferating cell nuclear antigen (PCNA) (DAKO, Kyoto, Japan).
Determination of sulfated glycosaminoglycan synthesis
Synthesis of sulfated glycosaminoglycans by BVMs was measured by
[35S] incorporation as described previously
(Ohta et al., 1999). Briefly,
we measured glycosaminoglycan synthesis at 24-48 hours and 48-72 hours after
the start of culture. For the measurement of glycosaminoglycan synthesis at
24-48 hours, BVMs were cultured for 24 hours with DMEM supplemented with 5%
dialyzed FCS and then incubated in medium supplemented with 0.5% dialyzed FCS
containing 45 mCi/ml of [35S] sulfate (Dupont NEN Research
Products, Boston, MA) for 24 hours. In the measurement of glycosaminoglycan
synthesis at 48-72 hours, BVMs were cultured for 24 hours with DMEM
supplemented with 5% dialyzed FCS and incubated in medium supplemented with
0.5% dialyzed FCS for 24 hours. Then, these were incubated in medium
supplemented with 0.5% dialyzed FCS containing 45 mCi/ml of [35S]
sulfate for 24 hours. After removal of the medium and washing with PBS five
times, BVMs were incubated with 0.5 ml of 0.25% trypsin and 0.1% collagenase
at 37°C for 30 minutes, and the resultant cell suspension was collected.
The dishes were washed with 0.5 ml of PBS. The cell suspension and the washing
solution were combined, and the cells were removed by centrifugation. Aliquots
of 0.4 ml of supernatant from each BVM were digested with 1 mg of pronase
(Wako Chemical Co.) for 3 hours at 37°C. The digests were mixed with 0.1
ml of 0.2 M NaCl containing 2 mg of chondroitin 4-sulfate as a carrier and 0.5
ml of 1.0% cetylpyridinium chloride (Wako Chemical Co.). Radioactivity in the
precipitated cetylpyridinium-glycosaminoglycan complex was measured by liquid
scintillation counting as described previously
(Ohta et al., 1999
). To
determine incorporation into chondroitin sulfates or heparan sulfate, the
pronase digests were boiled for 10 minutes and digested with 0.35 units of
chondroitinase ABC or a mixture of 30 milliunits each of heparitinase I and
II. The reduced radioactivity recovered in the
cetylpyridinium-glycosaminoglycan complex owing to enzymatic digestion was
assigned to be in the glycosaminoglycan.
Estimation of MK receptors and PG-M/versican by western blotting
analysis or by reverse transcription polymerase chain reaction
HUVEC or HASMC were plated at a density of 1.0x106 cells
in a 10 cm diameter dishes with each growth medium. On the following day,
these were incubated with serum-free DMEM with or without MK (100 ng/ml) for
24 hours at 37°C. Cells were washed twice with PBS and were collected by
scraping. BVMs cultured for 3 days with or without MK was also used as
samples.
Proteins were separated by 7% SDS-PAGE, and PTP and LRP were detected
by western blotting as described previously
(Muramatsu et al., 1993
) with
anti-human PTP
antibody or antimouse LRP antibody. The immunoreactive
bands were revealed by an enhanced chemiluminescence kit (Amersham Life
Science, Buchinghamshire, England). For quantitative estimation, bands were
scanned by Chemi Doc (Bio-Rad, Tokyo, Japan).
Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) of PG-M/versican was performed by using the one step RT-PCR kit (QIAGEN Gmbh., Hilden, Germany). For these reactions, 1.0x105 cells were used. The oligonucleotides used for amplification of human PG-M/versican cDNA fragment (nucleotide number 5377-6061, GenBank accession number X15998) were as follows: forward, CATCCCTGCCAATTCCTC, and reverse, TCTGTGGGAGAAGCTTCC. They were heated at 37°C for 30 minutes and at 94°C for 3 minutes, and then subjected to 35 cycles of denaturation (94°C for 30 seconds), annealing (55°C for 30 seconds) and extension (72°C for 30 seconds). Amplification products were subjected to electrophoresis in 1.5% agarose gel and stained with ethidium bromide. For quantitative estimation, bands were scanned by Chemi Doc.
ELISA
The amounts of IL-8, HGF, bFGF and VEGF were determined by ELISA with
commercial kits according to the manufacturer's instructions, whereas that of
MK was determined as described previously
(Muramatsu et al., 1996).
Statistical analysis
The values are expressed as means±s.d. Group means were compared
using the two-tailed Student's t-test. Differences were analyzed
statistically by ANOVA. Values with P<0.05 were considered
significant.
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Results |
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To determine whether the increase in the number of HUVEC in BVMs by MK was caused by an increased cell proliferatin, we stained the BVM sections with anti-PCNA antibody, which stains the nuclei of dividing cells. On Day 1, the numbers of intensely stained HUVEC were much higher in BVMs treated with MK as compared with control BVMs (Fig. 2). On Day 3, numbers of proliferating HUVEC decreased both in control BVMs and in BVMs treated with MK.
|
In relation to secretion of extracellular matrix, we assayed glycosaminoglycan synthesis by determining incorporation of [35S]O4 into glycosaminoglycans. When MK was added to HASMC gel and HUVEC cultured separately, we observed no enhancement of glycosaminoglycan synthesis. In BVMs, MK enhanced glycosaminoglycan synthesis by 1.68-fold at the dose of 50 ng/ml and 2.52-fold at 100 ng/ml, but was less effective at 10 ng/ml (Fig. 3). The ratio of radioactive sulfate incorporation into chondroitin sulfate and heparan sulfate in BVMs was 1: 0.6, and the value was not changed after MK treatment; MK stimulated the synthesis of both chondroitin sulfate and heparan sulfate.
|
To examine the effects of increased synthesis of glycosaminoglycans, we also added chondroitin-4 sulfate, chondroitin sulfate E, dermatan sulfate and heparin at the concentration of 10 µg/ml to the culture of BVMs together with 100 ng/ml of MK. However, the action of MK was not significantly affected (data not shown).
Role of HASMC in the MK-dependent increase in proliferation of
HUVEC
When MK (100 ng/ml) was added to HUVEC or HASMC cultured separately, no
increase in cell proliferation was observed
(Fig. 4A). However, when HUVEC
were co-cultured with HASMC at a ration of 1:10, respectively, MK
significantly increased the cell proliferation
(Fig. 4A). Poly-L-lysine showed
no effects. When conditioned medium of HASMC cultured with 100 ng/ml MK was
added, proliferation of HUVEC was increased; conditioned medium of HASMC
cultured without MK was slightly active on Day 1, but was not active on Day 2
(Fig. 4B). Thus, co-operation
with HASMC is required for stimulation of HUVEC proliferation by MK.
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Identification of IL-8 as a factor secreted by HASMC
The effectiveness of HASMC-conditioned medium suggested that a factor
secreted by HASMC in the presence of MK promoted proliferation of HUVEC. To
identify such a factor in the conditioned medium of HASMC, we performed an
ELISA for three human growth factors, that is, VEGF, HGF and bFGF, as wells as
the cytokine IL-8. The level of IL-8 was significantly altered by treatment
with MK for 24 hours; 10 ng/ml of MK caused a 2.39-fold increase of IL-8 level
in the conditioned medium (Table
2). The levels of other factors were not significantly altered by
MK.
|
To determine whether IL-8 is a mediator of the actions of MK, we added anti-IL-8 antibody to the culture of BVMs and found that the effect of MK was suppressed on Day 3 (Fig. 1H, Table 1). Various concentrations of IL-8 were also added to BVMs. In this experiment, BVMs were observed only on day 1 and day 2, since culture in DMEM without serum was required to examine the effects of IL-8, and this caused damage to BVMs on day 3. On day 2, IL-8 at 1000 ng/ml induced stratification of endothelial cells to a degree similar to that induced by 100 ng/ml MK. Lower concentrations of IL-8 were less effective (Fig. 5). In the serum-free system, anti-IL-8 antibody also inhibited MK action (Fig. 5).
|
Expression of MK and MK receptors in HUVEC and HASMC
To understand the basis of MK-dependent interactions between HUVEC and
HASMC, we investigated whether MK and MK receptors are expressed in these
cells. ELISA revealed that the HUVEC-conditioned medium, which was collected
20 hours after initiation of culture, had 0.9 ng/ml of MK, whereas no MK (less
than 0.05 ng/ml) was detected in the conditioned medium of HASMC.
Previous studies have indicated that the MK receptor is a molecular complex
containing LRP (Muramatsu et al.,
2000) and proteoglycans such as PTP
(Maeda et al., 1999
).
PTP
was detected both in HUVEC and HASMC, and its amount increased in
both cells and also in BVMs after MK treatment
(Fig. 6A). The amount of LRP
was below detection in cells before MK treatment, whereas it became detectable
after MK treatment in HASMC but not in HUVEC
(Fig. 6A). The level of
expression of PG-M/versican, which is a pericellular chondroitin sulfate
proteoglycan and binds to MK (Zou et al.,
2000
), was increased less significantly after treatment with MK
(Fig. 6B). The result with the
induction of LRP in HASMC is consistent with the view that HASMC are the
target of MK.
|
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Discussion |
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MK was identified as an angiogenic factor, on the basis of its in vivo
angiogenic activity of cells transfected with MK cDNA
(Choundhuri et al., 1997).
However, our repeated attempts to demonstrate in vitro angiogenic activity of
MK failed (Y.S., H.M., Y.T., K.-I.H. et al., unpublished). Proliferation of
endothelial cells is an important step in angiogenesis. The present results
provided in vitro evidence for the reported in vivo angiogenic activity of MK
for the first time. Furthermore, the requirement of the presence of smooth
muscle cells for the effect of MK on HUVEC explains the failure to observe
direct MK activity on endothelial cells. MK is overexpressed in a number of
human carcinomas, namely, gastric, colon and hepatocellular carcinomas
(Tsutsui et al., 1993
;
Aridome et al., 1995
;
Ye et al., 1999
), breast
carcinoma (Garver et al.,
1994
), lung carcinoma (Garver
et al., 1993
), urinary bladder carcinoma
(O'Brien et al., 1996
) and
neuroblastoma (Nakagawara et al.,
1995
). This enhanced expression of MK implies that it is
beneficial to tumor growth. MK enhances cell growth
(Muramatsu and Muramatsu,
1991
) and cell migration (Takada et al., 1996;
Maeda et al., 1999
;
Horiba et al., 2000
;
Qi et al., 2001
) and
suppresses apoptosis (Owada et al.,
1999
; Qi et al.,
2000
). Together with the above-mentioned activities, which will
help tumor growth and spread, the angiogenic activity of MK will significantly
contribute to tumor progression. Means to suppress MK expression or MK
activity in tumor tissue should be explored with the aim of development of new
treatment methods for malignancy.
Although proteoglycans play essential roles in MK signaling
(Kaneda et al., 1996;
Ahkter et al., 1998
;
Maeda et al., 1999
;
Ueoka et al., 2000
),
glycosaminoglycans including heparin did not inhibit MK activity to BVMs.
Previously, we observed that heparin did not inhibit MK action on bovine
aortic endothelial cells to enhance fibrinolytic activity, instead
heparitinase digestion of these target cells abolished the MK activity
(Ahkter et al., 1998
). Most
probably, endothelial cells have high-affinity binding sites for
glycosaminoglycans and prevent the action of glycosaminoglycans to inhibit MK
activity.
In enhancing HUVEC proliferation, the direct target of MK was identified as
HASMC, which secreted factor(s) acting on HUVEC. Furthermore, LRP, a component
of the MK receptor (Muramatsu et al.,
2000) was upregulated in HASMC. We also noticed that HUVEC
produced MK, whereas HASMC did not. These findings place MK as a factor
involved in epithelial-mesenchymal interactions, as has been suggested by
earlier studies (Kadomatsu et al.,
1990
; Mitsiadis et al.,
1995a
; Mitsiadis et al.,
1995b
; Toriyama et al., 1996). In tissues undergoing
epithelial-mesenshymal interactions, MK is generally expressed more strongly
in epithelial cells (Kadomatsu et al.,
1990
; Mitsiadis et al.,
1995b
). However, MK often acts on mesenchyme-derived cells; it
enhances growth of NIH3T3 cells (Muramatsu
and Muramatsu, 1991
), synthesis of extracellular matrix molecules
by dermal fibroblasts (Yamada et al.,
1997
) and fibroblast-mediated collagen gel contraction
(Sumi et al., 2000
). HASMC
studied here are also cells derived from the mesenchyme. Therefore, MK might
play a significant role in the complex interplay of the epithelial and
mesenchymal cell layers.
We found that MK increased IL-8 production by HASMC. IL-8 belongs to a
family of small, structurally related cytokines similar to platelet factor 4
(Clark-Lewis et al., 1993). It
is produced by phagocytes and mesenchymal cells exposed to inflammatory
stimuli (interleukin-1 or tumor necrosis factor) and activates neutrophils,
inducing chemotaxis and exocytosis. IL-8 promotes HUVEC growth and migration,
which is similar to bFGF, and induces corneal neovascularization
(Strieter et al., 1992
). Thus,
IL-8 was expected to be a mediator of the action of MK on endothelial cells.
Indeed, anti-IL-8 antibody inhibited the action of MK on BVMs. Furthermore,
addition of 1000 ng/ml IL-8 to BVMs resulted in stratification of HUVEC to the
same degree as observed upon addition of 100 ng/ml MK. We noticed that the
level of IL-8 in culture medium of MK-treated HASMC was only around 20 ng/ml.
IL-8, which was added to the medium of BVMs at the level of 100 ng/ml, showed
little effect. In BVMs treated with MK, IL-8 is delivered from the basal layer
facing the collagen gel, and this difference in delivery may partly explain
the ineffectiveness of exogenously added IL-8 at 100 ng/ml. However, the
difference may be better explained by the presence of other factors that
enhance the action of IL-8. It is even possible that upregulation of such a
factor is another critical event.
Recently, we found that MK increases expression of MIP-2 in proximal
tubular epithelial cells (Sato et al.,
2001). MIP-2 is probably the mouse counterpart of IL-8. The
increased MIP-2 level and the direct chemotactic activity of MK on neutrophils
explain MK-dependent recruitment of neutrophils to injured renal tubules
(Sato et al., 2001
). The
increased expression of IL-8/MIP-2 by MK in two entirely different systems
suggested that IL-8/MIP-2 is an important mediator of MK action at the tissue
level.
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Acknowledgments |
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Owada, K., Sanjo, N., Kobayashi, T., Mizusawa, H., Muramatsu, H., Muramatsu, T. and Michikawa, M. (1999). Midkine inhibits caspase-dependent apoptosis via the activation of mitogen-activated protein kinase and phosphatidyl-inositol 3-kinase in cultured neurons. J. Neurochem. 73,2084 -2092.[Medline]
Qi, M., Ikematus, S., Ichihara-Tanaka, K., Sakuma, S., Muramatsu, T. and Kadomatsu, K. (2000). Midkine rescues Wilms' tumor cells from cisplatin-induced apoptpsis: regulation of Bcl-2 expression by midkine. J. Biochem. 127,269 -277.[Abstract]
Qi, M., Ikematsu, S., Maeda, N., Ichihara-Tanaka, K., Sakuma,
S., Noda, M., Muramatsu, T. and Kadomatsu, K. (2001).
Haptotactic migration induced by midkine: Involvement of protein-tyrosine
phosphatase, mitogen-activated protein kinase and phosphatidylinositol
3-kinase. J. Biol. Chem.
276,15868
-15875.
Salama, R. H. M., Muramatsu, H., Zou, K., Inui, T., Kimura, T. and Muramatsu, T. (2001). Midkine binds to 37-kDa laminin protein precursor, leading to nuclear transport of the complex. Exp. Cell Res. 270,13 -20.[Medline]
Sato, W., Kadomatsu, K., Yuzawa, Y., Muramatsu, H., Hotta, N.,
Matsuo, S. and Muramatsu, T. (2001). Midkine is involved in
neutrophil infiltration into the tubulointerstitium in ischemic renal injury.
J. Immunol. 167,3463
-3469.
Strieter, R. M., Kunkel, S. L., Elner, V. M., Martonyi, C. L., Koch, A. E., Polverini, P. J. and Elner, S. G. (1992). Interleukin-8: A corneal factor that induces neovascularization. Am. J. Pathol. 141,1279 -1284.[Abstract]
Sumi, Y., Muramatsu, H., Hata, K. I., Ueda, M. and Muramatsu, T. (2000). Midkine enhances early stages of collagen gel contraction. J. Biochem. 127,247 -251.[Abstract]
Takada, T., Toriyama, K., Muramatsu, H., Song, X.-J., Torii, S. and Muramatsu, T. (1997). Midkine, a retinoic acid-inducible heparin-binding cytokine in inflammatory responses: chemotactic activity to neutrophils and association with inflammatory synovitis. J. Biochem. 122,453 -458.[Abstract]
Tomomura, M., Kadomatsu, K., Matsubara, S. and Muramatsu, T. (1990). A retinoic acid-responsive gene, MK, found in the teratocarcinoma system. Heterogeneity of the transcript and the nature of the translation product. J. Biol. Chem. 295,10765 -10770.
Toriyama, K., Muramatsu, H., Hoshino, T., Torii, S. and Muramatsu, T. (1997). Evaluation of heparin-binding growth factors in rescueing morphogenesis of heparitinase-treated mouse embryonic lung explants. Differentiation 61,161 -167.[Medline]
Tsutsui, J., Kadomatsu, K., Matsubara, S., Nakagawara, A., Hamanoue, M., Takao, S., Shimazu, H., Ohi, Y. and Muramatsu, T. (1993). A new family of heparin-binding growth differentiation factors: increased midkine expression in Wilms' tumor and other human carcinomas. Cancer Res. 53,1281 -1285.[Abstract]
Ueoka, C., Kaneda, N., Okazaki, I., Nadanaka, S., Muramatsu, T.
and Sugahara, K. (2000). Neuronal cell adhesion mediated by
the heparin-binding neuroregulatory factor, midkine, is specifically inhibited
by chondroitin sulfate E. Structural and functional implication of the
oversulfated chondroitin sulfate. J. Biol. Chem.
275,37407
-37413.
Yamada, H., Inazumi, T., Tajima, S., Muramatsu, H. and Muramatsu, T. (1997). Stimulation of collagen expression and glycosaminoglycan synthesis by midkine in human skin fibroblasts. Arch. Dermatol. Res. 289,429 -433.[Medline]
Ye, C., Fan, Q.-W., Akiyama, S., Kasai, Y., Matsuyama, M., Muramatsu, T. and Kadomatsu, K. (1999). Expression of midkine in the early stage of carcinogenesis in human colorectal cancer. Br. J. Cancer. 79,179 -184.[Medline]
Zou, K., Muramatsu, H., Ikematsu, S., Sakuma, S., Salama, R. H.,
Shinomura, T., Kimata, K. and Muramatsu, T. (2000). A
heparin-binding growth factor, midkine, binds to a chondroitin sulfate
proteoglycan, PG-M/versican. Eur. J. Biochem.
267,4046
-4053.