© 1995 American Heart Association, Inc.
Articles |
Increased Mitogenic Response to Heparin-Binding Epidermal Growth Factor–like Growth Factor in Vascular Smooth Muscle Cells of Diabetic Rats
From the Second Department of Internal Medicine and Department of Biochemistry (S.H., N.T.), Osaka University Medical School, Osaka, Japan.
Correspondence to Kazuto Fukuda, MD, Second Department of Internal Medicine, or Shigeki Higashiyama, MD, Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita 565, Japan.
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Abstract We investigated the mitogenic effects of heparin-binding epidermal growth factor–like growth factor (HB-EGF) in vascular smooth muscle cells (SMCs) obtained from rats with streptozotocin (STZ)-induced diabetes and evaluated the role of heparan sulfate proteoglycan (HSPG) in inducing these effects. HB-EGF significantly increased DNA synthesis in the SMCs of diabetic rats (STZ-SMCs) compared with control rats (control SMCs). However, the mitogenic effects of EGF, which shares EGF receptors with HB-EGF, and basic fibroblast growth factor, another heparin-binding growth factor, were similar in STZ-SMCs and control SMCs. The mitogenic response to HB-EGF in SMCs of insulin-treated diabetic rats was similar to the response in control SMCs. HB-EGF–induced autophosphorylation of EGF receptors was increased in STZ-SMCs compared with control SMCs, although the number of EGF receptors in STZ-SMCs was 40% of that in controls. This increased mitogenic response to HB-EGF in STZ-SMCs was completely inhibited by treatment with heparitinase, chlorate, and a synthetic peptide corresponding to the heparin-binding domain of HB-EGF. Compared with heparan sulfate isolated from control SMCs, heparan sulfate isolated from STZ-SMCs was of smaller molecular size and caused a greater mitogenic effect of HB-EGF. These findings suggest that the mitogenic response to HB-EGF is increased in SMCs of diabetic rats. Changes in cell-associated heparan sulfate in STZ-SMCs may be related to the increased mitogenic response to HB-EGF.
Key Words: HB-EGF • vascular smooth muscle cells • heparan sulfate proteoglycan • diabetes mellitus
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Introduction |
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Diabetes mellitus is a major risk factor for atherosclerosis.1 2 3 Hyperglycemia,3 hyperlipidemia,4 hyperinsulinemia,5 enhanced platelet aggregation,6 and elevated concentrations of lipid peroxides7 are all believed to contribute to diabetic macroangiopathy. Because SMC proliferation and migration are essential components of these processes,8 a fundamental understanding of the mechanisms of SMC proliferation and migration would greatly facilitate the development of effective strategies for preventing diabetes-induced atherosclerosis. Although cultured aortic SMCs from STZ-induced diabetic rats and alloxan-induced diabetic rabbits have exhibited faster growth than normal SMCs,9 10 11 the biologic mechanisms of the growth of SMCs in the diabetic state have not been clarified.
HB-EGF, a protein secreted by the human macrophage-like
cell line U-937, is a potent mitogenic and migratory factor
for SMCs.12 13 14 15 Mature bioactive HB-EGF is a 22-kD
glycoprotein that binds to the EGF receptor, stimulating
phosphorylation. The primary sequence of HB-EGF
contains a COOH-terminal portion that is 40% structurally identical to
EGF and to TGF-
and an NH2-terminal extension region
that is not present in EGF or TGF-.12 13 HB-EGF is
synthesized by macrophages, monocytes,16 vascular
endothelial cells,17 18
keratinocytes,19 and SMCs.20
Previously we have observed the increased expression of HB-EGF in SMCs
and macrophages of human atherosclerotic
plaques.21 Lysophosphatidylcholine, a major component of
oxidized LDL, increases HB-EGF mRNA expression in human
monocytes16 and vascular endothelial
cells.17 Shear stress also upregulates the level of HB-EGF
mRNA in human umbilical vein endothelial
cells.18 Expression of HB-EGF mRNA in SMCs is induced by
platelet-derived growth factor (PDGF), b-FGF,
angiotensin II, and HB-EGF itself.20 These
findings suggest that HB-EGF may have a regulatory role in the growth
of SMCs in vivo.
HB-EGF demonstrates a more potent mitogenic effect on SMCs
than does EGF and TGF-, although all three growth factors bind to
the EGF receptor.12 The heparin-binding properties of
HB-EGF may facilitate its interaction with cell-surface HSPG, which
may modulate its biologic activity.12 13 22 Such
facilitation has been observed in the case of other heparin-binding
growth factors, such as b-FGF.
In the present study, we examined the mitogenic response of STZ-SMCs to HB-EGF in addition to the effect of HS synthesized by these SMCs on the mitogenic activity of HB-EGF.
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Methods |
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Growth Factors
Recombinant human HB-EGF was kindly provided by Dr Judith A. Abraham, Scios Nova Inc.23 Recombinant human EGF was purchased from Toyobo Co, Ltd. Recombinant human b-FGF was purchased from Austral Biologicals. The 125I-EGF (3.7 MBq/mL) used in our experiments was purchased from Amersham Life Science.
Animals
Diabetes was induced in male Wistar rats (250 g) by
administering intravenous injections of STZ (70 mg/kg)
(Wako). Beginning 3 days after these injections, half the diabetic rats
underwent a series of subcutaneous injections of protamine zinc insulin
(Novo Nordisc) administered daily at 5 PM for 4 weeks.
Age-matched control and diabetic rats were fed standard chow for 4
weeks. Fed rats were then anesthetized with sodium
pentobarbital (30 mg/kg) and exsanguinated via the abdominal aorta.
Serum and plasma samples were prepared from collected blood, and the
plasma concentration of glucose and the serum concentrations of
immunoreactive insulin, triglycerides, total
cholesterol, and free fatty acids were measured by
previously described methods.24 Diabetic rats were
characterized by significant hyperglycemia, hypoinsulinemia, and low
body weight. Serum free fatty acid, triglyceride, and total
cholesterol concentrations were significantly elevated in
diabetic rats compared with controls. Insulin treatment partially
normalized some of these biochemical parameters (Table 1
).
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Culture of SMCs
Rat aortic SMCs were isolated using the explant method of
Fischer-Dzoga et al.25 In brief, aortic explants were
obtained from the thoracic aorta and the adventitia was removed.
Explants were cultured in Dulbecco's modified Eagle medium (DMEM)
supplemented with 15% fetal bovine serum (Irvine Scientific), 100 U/mL
penicillin, and 100 µg/mL streptomycin at 37°C in 5%
CO2. After 2 weeks, the cells that had migrated out of the
explant were removed by trypsinization and seeded in T-75 flasks. Cells
were characterized as SMCs by morphological criteria (spindle shape and
hill-and-valley pattern) and by their expression of smooth
muscle -actin. SMCs were subcultured at a 1:4 split ratio
every week. Cells from the fourth to sixth passages were used in the
following experiments. Three different strains of control SMCs and
STZ-SMCs and two different strains of insulin-treated diabetic rat
SMCs were used, with similar results.
Effects of Growth Factors on DNA Synthesis in SMCs
DNA synthesis in SMCs was assessed by
[3H]thymidine incorporation. Cells were grown to
subconfluence (
30 000 to 40 000 cells/cm2) in 96-well
plates and placed in a growth-arresting medium consisting of DMEM
containing 0.1% bovine plasma-derived serum (0.1% PDS-DMEM) for
48 hours to induce a quiescent state. The medium was then replaced with
5% PDS-DMEM containing various concentrations of HB-EGF, EGF, or
b-FGF, and the cells were cultured for 24 hours. PDS, which is devoid
of platelet-derived mitogens, maintains SMCs in the quiescent
state for extended periods of time in culture.26 Twenty
hours after the addition of growth factors, cells were pulse labeled
with [3H]thymidine (37 kBq/well) (Amersham) for 4 hours.
Incorporated [3H]thymidine was counted via a beta plate
system (Pharmacia LKB).
EGF Receptor Binding Assay
Cells were washed twice with a binding buffer (DMEM, 50 mmol/L
BES, and 0.1% BSA) and incubated for 4 hours at 4°C in 24-well
plates with various amounts of 125I-EGF (3.7 MBq/mL) in the
presence or absence of a 100-fold excess of unlabeled
EGF.27 The cells were then washed four times with the
binding buffer and solubilized with 0.75 mL of a lysing buffer (0.01
mol/L Tris-HCl, 0.5% SDS, and 1 µmol/L EDTA). Cell-bound and
free 125I radioactivity was measured with a gamma counter.
Nonspecific binding to cells was usually 
10% of the total
binding. Data were applied to Scatchard plots using least-squares
analysis.
Autophosphorylation of the EGF
Receptor
Autophosphorylation of the EGF receptor was
assessed according to the method of Margolis et al.28
Quiescent SMCs were stimulated with 100 ng/mL HB-EGF for 10 minutes at
37°C. These cells were scraped into a lysis buffer containing 50
mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 10% glycerol, 1% Triton
X-100, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 10 µg/mL
leupeptin, 10 µg/mL aprotinin, 1 mmol/L PMSF, 200 µmol/L sodium
orthovanadate, 10 mmol/L sodium pyrophosphate, 100 mmol/L sodium
fluoride, and 30 mmol/L p-nitrophenyl phosphate.
After the protein content was measured according to the method of Lowry
et al,29 lysates of SMCs (100 µg protein) were
immunoprecipitated with sheep anti-human EGF receptor antibody (No.
06-129) (UBI) coupled to protein G–Sepharose (Sigma). After
immunoprecipitation, samples were washed with the lysis buffer, placed
in the sample buffer (25 mmol/L Tris-HCl, pH 6.8; 2% SDS; 10%
glycerol; and 0.05% bromophenol blue), and heated to 90°C for 5
minutes. Immunoprecipitated proteins were then separated on an SDS-gel
gradient (SPG-520L, ATTO) and transferred to polyvinylidene
difluoride membranes (Immobilon PVDF, Millipore).
Phosphorylation of EGF receptors on tyrosine residues
was analyzed by Western blot analysis by using rabbit
anti-phosphotyrosine antibodies (No. 05-321) (UBI) and subsequent
development with peroxidase-conjugated swine anti-rabbit IgG
antibodies (Dako). Visualization by fluorography with an enhanced
chemiluminescence system (Amersham) was then performed.
Heparitinase and Chlorate Treatment of Cells
To investigate the relationship between HSPG and the biologic
activity of HB-EGF, SMCs were treated as previously described with
heparitinase to digest cell-surface HS or with chlorate, an
inhibitor of HS sulfation.22 For heparitinase
treatment, SMCs were preincubated with 20 mU/mL heparitinase I (EC
4.2.2.8) (Sigma), beginning 1 hour before the addition of HB-EGF or
EGF. For chlorate treatment, SMCs were preincubated with 10 mmol/L
sodium chlorate (Aldrich) beginning 48 hours before the addition of
growth factors. The effects of growth factors on DNA synthesis were
investigated as described above.
Treatment of SMCs With a Synthetic Peptide Corresponding to the
Heparin-Binding Domain of HB-EGF
A synthetic peptide (P21) corresponding to the putative
21–amino acid heparin-binding sequence KRKKKGKGLGKKRDPCLRKYK of
human HB-EGF (amino acids 93 through 113 of the 208-residue
precursor)22 was synthesized by use of an Applied
Biosystems Peptide Synthesizer (courtesy of Margaret Ehrhardt [Brigham
and Women's Hospital, Boston, Mass]). Cells were preincubated with 25
µg/mL P21 beginning 1 hour before the addition of HB-EGF and b-FGF.
The DNA synthesis in SMCs was investigated as described above.
Isolation of Cell-Associated Protein-Free
Glycosaminoglycans
Cell-associated GAGs were isolated according to the method
of Schmidt and Buddecke.30 In brief, 10 quiescent cultures
(10-cm dishes) of SMCs were preincubated at 37°C for 48 hours in the
presence of [35S]sulfate (100 kBq/mL) (Amersham) or
D-[1,6-3H(N)]glucosamine
hydrochloride (93 kBq/mL) (New England Nuclear). Then the medium was
removed, and the cell layer was washed twice with PBS and trypsinized.
The trypsin digest was precipitated with 2.5-vol ethanol and potassium
acetate. Next, the precipitate was digested with 5 mg of crystallized
papain (Wako) in 50 mmol/L sodium acetate (pH 5.8), containing 10
mmol/L EDTA and 5 mmol/L cysteine, at 65°C for 24 hours. The
resulting digest was centrifuged, and the supernatant was
applied to a DEAE column (1 mL, DE 52, Whatman) equilibrated with 50
mmol/L Tris-HCl (pH 7.0), containing 1% Triton X-100 and preloaded
with 5 mg BSA. The column was washed with 3 vol of 50 mmol/L Tris-HCl,
containing 1% Triton X-100 and 3 vol of 50 mmol/L Tris-HCl, and then
eluted in a stepwise manner with 3 mL each of 0.1-mol/L and 0.7-mol/L
NaCl buffered with Tris-HCl. The 0.7-mol/L eluate (containing the total
GAGs) was digested with 10 mU/mL chondroitin ABC lyase (Seikagaku)
according to the method of Saito et al.31 The digest was
again subjected to DEAE chromatography under identical
conditions, and the HS fraction was obtained. Incorporation of
35S or 3H radioactivity in HS was determined by
scintigraphy. Cell protein contents per dish were
determined before trypsin digest.
Sephacryl S-200 Chromatography of HS
For determination of the relative molecular mass, isolated HS
was subjected to chromatography using a Sephacryl S-200
(Pharmacia) column (1x50 cm) equilibrated with 50 mmol/L sodium
acetate (pH 6.0), 50 mmol/L NaCl, and 0.2% SDS. After application of
the sample, the column was eluted with the same buffer at a flow rate
of 10 mL/h, and 0.5-mL fractions were collected. 35S
radioactivity in each fraction was determined using a scintillation
counter.
Effect of Cell-Associated HS on the Mitogenic Activity
of HB-EGF
Nonradiolabeled HS was isolated from quiescent control SMCs
(control-HS) and STZ-treated SMCs (STZ-HS). Control SMCs were treated
with 5 mmol/L chlorate for 48 hours or with 20 mU/mL heparitinase for 4
hours. Various concentrations of HS were then coincubated with HB-EGF
for 24 hours. DNA synthesis in SMCs was then determined.
Statistical Analysis
Data are presented as mean±SD. Differences between
group means were analyzed by the Student's t test
as indicated in the tables and figure legends. A value of
P<.05 was considered statistically significant.
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Results |
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Effects of Growth Factors on Proliferation of SMCs
In concentrations of up to 2.5 nmol/L, HB-EGF stimulated DNA synthesis in a dose-dependent manner in control SMCs, STZ-SMCs, and SMCs of insulin-treated diabetic rats (insulin-treated STZ-SMCs) (Fig 1
). The mitogenic response to
HB-EGF was significantly higher in STZ-SMCs than in control SMCs.
Insulin treatment inhibited this increase in the mitogenic
response in STZ-SMCs. The response to EGF was almost similar in all
three groups (Fig 1), and the mitogenic effect of HB-EGF in
STZ-SMCs was significantly more potent than the effect of EGF. The
mitogenic response to b-FGF was almost similar in all three
groups (Fig 1). We tested three growth factors at concentrations up to
10 nmol/L. In higher concentrations than 2.5 nmol/L, the response curve
did not show dose dependence. Sometimes, the response at 10 nmol/L was
lower than that at 2.5 nmol/L or 0.62 nmol/L (data not shown).
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), STZ-induced
diabetic rats (
), and insulin-treated diabetic rats (
)
by incubation in DMEM containing 0.1% PDS for 48 hours. The
mitogenic response (DNA synthesis) was then assessed by
quantifying the [3H]thymidine incorporation in the
presence of various concentrations (0.01 to 2.5 nmol/L) of HB-EGF, EGF,
and b-FGF. Incorporation of [3H]thymidine is expressed as
the percent of the incorporation without growth factors (% of
control). Values represent the mean±SEM of triplicate
determinations.
EGF Receptor Analysis
Scatchard analysis demonstrated that the affinity of EGF
for EGF receptors was similar in control SMCs and STZ-SMCs
(Kd, 320 pmol/L and 300 pmol/L,
respectively). The number of receptors on the surfaces of STZ-SMCs was
40% of the number on control SMCs (1.26x104/cells
versus 3.13x104/cells) (Fig 2).
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EGF Receptor Autophosphorylation
Western blot analysis demonstrated that HB-EGF–induced
autophosphorylation of the EGF receptor was
significantly increased in STZ-SMCs compared with control SMCs (Fig 3).
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Effects of Chlorate and Heparitinase on Stimulation of DNA
Synthesis by HB-EGF and EGF
The inhibitory effect of 10 mmol/L chlorate on
[3H]thymidine incorporation was significantly greater in
HB-EGF–treated cells than in EGF-treated cells (Fig 4).
In addition, the inhibitory effect of chlorate was more
pronounced in STZ-SMCs than in control SMCs (85% inhibition in
STZ-SMCs versus 65% in control SMCs). Similarly, 20 mU/mL heparitinase
caused greater inhibition of the mitogenic response to
HB-EGF in STZ-SMCs than in control SMCs (70% in STZ-SMCs versus 50%
in control SMCs) (Fig 5). After chlorate and
heparitinase treatment, the response to HB-EGF was similar in control
SMCs and STZ-SMCs.
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Effects of a Synthetic Peptide Corresponding to the Heparin-Binding
Domain of HB-EGF on DNA Synthesis Stimulated by HB-EGF and
b-FGF
The synthetic peptide P21 inhibited the mitogenic
response to HB-EGF more potently in STZ-SMCs than in control SMCs (50%
inhibition versus 20% inhibition) (Fig 6). After P21
treatment, the response to HB-EGF was similar in STZ-SMCs and control
SMCs. P21 did not alter the mitogenic activity of
b-FGF.
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Cell-Associated HS in Control SMCs and STZ-SMCs
Incorporation of 35S and 3H in HS was 70%
lower in STZ-SMCs than in control SMCs. The
35S/3H ratio for HS was similar in control SMCs
and STZ-SMCs (Table 2). The partition coefficient (Kav)
of elution maxima of HS from STZ-SMCs was higher than that of HS from
control SMCs (Kav, 0.23 in STZ-SMCs versus Kav, 0.11 in control SMCs)
(Fig 7).
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Effect of HS on Bioactivity of HB-EGF in SMCs
HS isolated from SMCs restored the mitogenic response
to HB-EGF in SMCs treated with chlorate in a dose-dependent manner.
STZ-HS was approximately twice as potent as control HS for equivalent
amounts of cell protein (Fig 8). STZ-HS also increased
the response to HB-EGF in SMCs treated with heparitinase in the same
manner (data not shown). HS did not affect the basal
[3H]thymidine incorporation in quiescent SMCs.
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Discussion |
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In the present study, we demonstrate that the mitogenic effect of HB-EGF was significantly increased in STZ-SMCs compared with control SMCs. However, the mitogenic effects of EGF, which shares EGF receptors with HB-EGF, and b-FGF, another heparin-binding growth factor, were similar in STZ-SMCs and control SMCs. The mitogenic response to HB-EGF in SMCs of insulin-treated diabetic rats was similar to the response in control SMCs. HB-EGF is a potent mitogenic and migratory factor for SMCs that is equal to PDGF12 13 14 15 22 and is synthesized by macrophages, monocytes,16 vascular endothelial cells,17 18 and SMCs.20 Thus, an increase in the responsiveness to HB-EGF may be involved in the pathogenesis of diabetic vascular changes.
HB-EGF–induced autophosphorylation of EGF receptors was significantly increased in STZ-SMCs, although the number of EGF receptors was decreased to only 40% of the control number. This increased mitogenic response to HB-EGF in STZ-SMCs was completely inhibited by treatment with heparitinase, chlorate, and a synthetic peptide corresponding to the heparin-binding domain of HB-EGF. HS isolated from STZ-SMCs enhanced the bioactivity of HB-EGF to a greater degree than HS isolated from control SMCs. These findings suggest that the increased responsiveness to HB-EGF in STZ-SMCs may have been related to alterations of cell-surface HS chains rather than to changes in the EGF receptor itself.
Previous studies have shown that the biologic activities of b-FGF, vascular endothelial growth factor, and HB-EGF, which are all heparin-binding growth factors, depend on a dual receptor system consisting of a high-affinity receptor and a cell-surface-specific HSPG.32 33 34 35 Possible mechanisms of the activity of HSPG in the regulation of growth factor activity are (1) HSPG concentrates heparin-binding growth factors on the cell surface, making them more readily available to the cell,36 (2) HSPG stabilizes or alters the conformation of heparin-binding growth factors and/or their receptors, thereby increasing ligand-receptor affinity,34 35 (3) HSPG stabilizes dimerization of heparin-binding growth factors, which in turn facilitates receptor dimerization,37 38 and (4) HSPG lowers the off-rate component of binding to high-affinity receptors by heparin-binding growth factors.39
Several species of HSPG, such as syndecan, fibroglycan, and glypican, fail to promote high-affinity receptor binding of b-FGF, although total cell-derived HSPG promotes such binding.40 This fact suggests that a unique species of HSPG may be involved in the regulation of receptor binding and the biologic activity of b-FGF. A recent study has identified perlecan as a major candidate for a b-FGF low-affinity receptor.41 In this study, P21, a synthetic peptide corresponding to the heparin-binding domain of HB-EGF, inhibited HB-EGF–mediated effects, but not b-FGF–mediated effects, suggesting that the HSPG species that corresponds to the heparin-binding domain of HB-EGF may differ from the species that corresponds to the domain of b-FGF. Therefore, changes in HS of STZ-SMCs may selectively correspond to the heparin-binding domain of HB-EGF, thus stimulating the bioactivity of HB-EGF.
The amount of cell-associated HSPG determined by the incorporation of [35S]sulfate or [3H]glucosamine was significantly decreased in STZ-SMCs compared with control SMCs. In addition, the relative molecular mass of HS isolated from STZ-SMCs was smaller than that of HS isolated from control SMCs. These findings suggest that the HS in STZ-SMCs is qualitatively different from that in control SMCs. However, the mechanism whereby altered HS in STZ-SMCs potentiated the mitogenic effect of HB-EGF was not clarified in this study. It is not clear which species of HSPG stimulates ligand-receptor binding, thus regulating the bioactivity of HB-EGF. Further studies are needed to clarify the differences between cell-associated HS in control SMCs and STZ-SMCs and the detailed characterization of HS that mediates the bioactivity of HB-EGF.
The increased mitogenic effect of HB-EGF in SMCs of
diabetic rats was normalized by insulin treatment, suggesting that the
effect was caused by metabolic changes induced by an
insulin deficiency. Several studies have shown that HSPG synthesis is
altered in the diabetic state in various types of cells. Among the
metabolic changes observed in diabetes, hyperglycemia has
been thought to be a possible factor causing the alteration of HSPG
synthesis. High doses of glucose have been found to alter proteoglycan
synthesis in avian cartilage cells.42 High-dose
glucose has also been observed to reduce HSPG synthesis in
mesangial cells, but the mechanism of this effect remains
unclear.43 44 In SMCs, HSPG synthesis is altered by
cytokines such as tumor necrosis factor–45 or
by a proliferating state.30 However, HSPG synthesis in
SMCs in the diabetic state have not been well examined. Therefore, we
are currently investigating regulation of HSPG synthesis in SMCs by
glucose and insulin.
The increased mitogenic response to HB-EGF in STZ-SMCs persisted during passages up to the sixth, indicating that the change was not a transient response to alterations in the levels of glucose, insulin, triglycerides, or other factors. Stability in the presence of passaging suggests a genetic difference between STZ-SMCs and control SMCs. We might have obtained a different subpopulation of SMCs from the STZ-treated and control rat aortas.
In this study, we observed no SMC hyperplasia or migration into the intima in our STZ-induced diabetic rats, which are common phenomena in atherosclerotic lesions in the human aorta.8 It remains to be clarified whether factors other than HB-EGF are required to induce such atherosclerotic lesions in STZ-treated rats or whether the absence of such lesions in the present study was related to the relatively short experimental period. Further study is needed to determine whether similar alterations in responsiveness to HB-EGF occur in SMCs derived from atherosclerotic lesions.
In conclusion, our results demonstrate that the mitogenic responsiveness to HB-EGF is significantly increased in STZ-SMCs, possibly because of changes in the cell-surface HS.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported in part by a grant-in-aid for cancer research to Drs Higashiyama and Taniguchi (No. 05151047), a grant-in-aid to Dr Matsuzawa (No. 04404085), and a grant-in-aid to Dr Kawata (No. 06557058) from the Ministry of Education, Science, and Culture of Japan. Dr Higashiyama is the recipient of a Searl Scientific Research Fellowship and a Suzuken Memorial Foundation Fellowship. We are grateful to Dr Judith A. Abraham for her generous gift of recombinant human HB-EGF.
Received May 2, 1995; accepted July 19, 1995.
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References |
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1. Santen RJ, Willis PW, Fajans SS. Atherosclerosis in diabetes mellitus: correlation with serum lipid levels, adiposity and serum insulin level. Arch Intern Med. 1972;130:833-843.
2.
Kannel WB, McGee DL. Diabetes and
cardiovascular disease: the Framingham Study.
JAMA. 1979;241:2035-2038.
3. Keen H, Jarrett RJ, Fuller JH, McCartney P. Hyperglycemia and arterial disease. Diabetes. 1981;30(suppl 2):49-53.
4. Kannel WB, Castelli WP, Gordon T, Macnamara PM. Serum cholesterol, lipoproteins and the risk of coronary heart disease. Ann Intern Med. 1971;74:1-12.
5. Stout RW. Insulin-stimulated lipogenesis in arterial tissue in relation to diabetes and atheroma. Lancet. 1968;2:702-703. [Medline] [Order article via Infotrieve]
6. Bern MM. Platelet functions in diabetes mellitus. Diabetes. 1978;27:342-350. [Medline] [Order article via Infotrieve]
7. Sato Y, Hotta N, Sakamoto N, Matsuoka S, Ohishi N, Yagi K. Lipid peroxide level in plasma of diabetic patients. Biochem Med. 1979;21:104-107. [Medline] [Order article via Infotrieve]
8. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801-809. [Medline] [Order article via Infotrieve]
9. Hauss WH, Denes R, Mey J, Lehmann R. Particularities of the vessel wall cells in experimental diabetes. Front Matrix Biol. 1979;7:183-192.
10. Alipui C, Tenner TE, Ramos K. Rabbit aortic smooth muscle cell culture: a model for the pharmacological study of diabetes-induced alterations in cell proliferation. J Pharmacol Methods. 1991;26:211-222. [Medline] [Order article via Infotrieve]
11. Kawano M, Koshikawa T, Kanzaki T, Morisaki N, Saito Y, Yoshida S. Diabetes mellitus induces accelerated growth of aortic smooth muscle cells: association with overexpression of PDGF ß-receptors. Eur J Clin Invest. 1993;23:84-90. [Medline] [Order article via Infotrieve]
12.
Higashiyama S, Abraham JA, Miller J, Fiddes JC,
Klagsburn M. A heparin-binding growth factor secreted by
macrophage-like cells that is related to EGF.
Science. 1991;251:936-939.
13.
Higashiyama S, Lau K, Besner G, Abraham JA, Klagsburn
M. Structure of heparin-binding EGF-like growth factor:
multiple forms, primary structure, and glycosylation of the mature
protein. J Biol Chem. 1992;267:6205-6212.
14. Besner G, Higashiyama S, Klagsburn M. Isolation and characterization of a macrophage-derived heparin-binding growth factor. Cell Regul. 1990;1:811-819. [Medline] [Order article via Infotrieve]
15. Abraham JA, Damm D, Bajardi A, Miller J, Klagsburn M, Ezekowitz AB. Heparin-binding EGF-like growth factor: characterization of rat and mouse cDNA clones, protein domain conservation across species, and transcript expression in tissues. Biochem Biophys Res Commun. 1993;190:125-133. [Medline] [Order article via Infotrieve]
16.
Nakano T, Raines EW, Abraham JA, Klagsburn M, Ross R.
Lysophosphatidylcholine upregulates the level of
heparin-binding epidermal growth factor-like growth factor mRNA
in human monocytes. Proc Natl Acad Sci U S A. 1994;91:1069-1073.
17. Kume N, Gimbrone MA Jr. Lysophosphatidylcholine transcriptionally induces growth factor gene expression in cultured human endothelial cells. J Clin Invest. 1994;93:907-911.
18. Morita T, Yoshizumi M, Kurihara H, Maemura K, Nagai R, Yazaki Y. Shear stress increases heparin-binding epidermal growth factor-like growth factor mRNA levels in human vascular endothelial cells. Biochem Biophys Res Commun. 1993;17:256-262.
19.
Hashimoto K, Higashiyama S, Asada H, Hashimura E,
Kobayashi T, Sudo K, Nakagawa T, Damm D, Yoshikawa K, Taniguchi N.
Heparin-binding epidermal growth factor-like growth
factor is an autocrine growth factor for human
keratinocytes. J Biol
Chem. 1994;269:20060-20066.
20.
Dluz SM, Higashiyama S, Damm D, Abraham JA, Klagsburn
M. Heparin-binding epidermal growth factor-like growth
factor expression in cultured fetal human vascular smooth muscle
cells. J Biol Chem. 1993;268:18330-18334.
21. Miyagawa J, Higashiyama S, Kawata S, Inui Y, Tamura S, Yamamoto K, Nishida M, Nakamura T, Yamashita S, Matsuzawa Y, Taniguchi N. Localization of heparin-binding EGF-like growth factor in the smooth muscle cells and macrophages of human atherosclerotic plaques. J Clin Invest. 1995;95:404-411.
22.
Higashiyama S, Abraham JA, Klagsburn M.
Heparin-binding EGF-like growth factor stimulation of smooth
muscle cell migration: dependence on interactions with cell surface
heparan sulfate. J Cell Biol. 1993;122:933-940.
23.
Thompson SA, Higashiyama S, Wood K, Pollitt NS, Damm D,
McEnroe G, Garrick B, Ashton N, Lau K, Hancock K, Klagsburn M, Abraham
JA. Characterization of sequences within heparin-binding
EGF-like growth factor that mediate interaction with heparin.
J Biol Chem. 1994;269:2541-2549.
24. Inui Y, Kawata S, Matsuzawa Y, Tokunaga K, Fujioka S, Tamura S, Kobatake T, Keno Y, Tarui S. Increased level of apolipoprotein B mRNA level in the liver of ventromedial hypothalamus lesioned obese rats. Biochem Biophys Res Commun. 1989;163:1107-1112. [Medline] [Order article via Infotrieve]
25. Fischer-Dzoga K, Jones RM, Vesslinovitch D, Wissler RW. Ultrastructural and immunohistochemical studies of primary cultures of aortic medial cells. Exp Mol Pathol. 1973;18:162-176. [Medline] [Order article via Infotrieve]
26.
Reilly C, McFall R. Platelet-derived
growth factor and transforming growth factor-ß regulate
plasminogen activator inhibitor-1
synthesis in vascular smooth muscle cells. J
Biol Chem. 1991;266:9419-9427.
27.
Epstein SE, Siegall CB, Biro S, Fu YM, FitzGerald D,
Pastan I. Cytotoxic effect of a recombinant chimeric toxin on
rapidly proliferating vascular smooth muscle cells.
Circulation. 1991;84:778-787.
28.
Margolis B, Beloot F, Honegger AM, Ullrich A,
Schlessinger J, Zilberstein A. Tyrosine kinase activity is
essential for the association of phospholipase C-
with the epidermal
growth factor receptor. Mol Cell Biol. 1990;10:435-441.
29.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ.
Protein measurement with the Folin phenol reagent.
J Biol Chem. 1951;193:265-275.
30. Schmidt A, Buddecke E. Changes in heparan sulfate structure during transition from the proliferating to the non-dividing state of cultured arterial smooth muscle cells. Eur J Cell Biol. 1990;52:229-235.[Medline] [Order article via Infotrieve]
31.
Saito H, Yamagata T, Suzuki S. Enzymatic methods
for the determination of small quantities of isomeric chondroitin
sulfates. J Biol Chem. 1968;243:1536-1542.
32.
Rapraeger AC, Krufka A, Olwin BB. Requirement of
heparan sulfate for b-FGF-mediated fibroblast growth and myoblast
differentiation. Science. 1991;252:1705-1708.
33.
Gitay-Goren H, Soker S, Vlodavsky I, Neufeld G.
The binding of vascular endothelial growth
factor to its receptors is dependent on cell surface-associated
heparin-like molecules. J Biol Chem. 1992;267:6093-6098.
34. Klagsburn M, Baird A. A dual receptor system is required for basic fibroblast growth factor activity. Cell. 1991;67:229-231. [Medline] [Order article via Infotrieve]
35. Yayon A, Klagsburn M, Esko JD, Leder P, Ornitz DM. Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell. 1991;64:841-848. [Medline] [Order article via Infotrieve]
36. Klagsburn M. The affinity of fibroblast growth factors (FGF) for heparin: FGF-heparan sulfate interaction in cells and extracellular matrix. Curr Opin Cell Biol. 1990;2:857-863. [Medline] [Order article via Infotrieve]
37. Spivak-Kroizman T, Lemmon MA, Dikic I, Ladbury JE, Pinchas D, Huang J, Jaye M, Crumley G, Schlessinger J, Lax I. Heparin-induced oligomerization of FGF molecules is responsible for FGF receptor dimerization, activation, and cell proliferation. Cell. 1994;79:1015-1024. [Medline] [Order article via Infotrieve]
38.
Ornitz DM, Yayon A, Flanagan JG, Svahn CM, Levi E,
Leder P. Heparin is required for cell-free binding of basic
fibroblast growth factor to a soluble receptor and for mitogenesis in
whole cells. Mol Cell Biol. 1992;12:240-247.
39. Nugent MA, Edelman ER. Kinetics of basic fibroblast growth factor binding to its receptor and heparan sulfate proteoglycan: a mechanism for cooperativity. Biochemistry. 1992;31:8876-8883. [Medline] [Order article via Infotrieve]
40.
Aviezer D, Levy E, Safran M, Svahn C, Buddecke E,
Schmidt A, David G, Vlodavsky I, Yayon A. Differential
structural requirements of heparin and heparan sulfate proteoglycans
that promote binding of basic fibroblast growth factor to its
receptor. J Biol Chem. 1994;269:114-121.
41. Aviezer D, Hecht D, Safran M, Eisinger M, David G, Yayon A. Perlecan, basal lamina proteoglycan, promotes basic fibroblast growth factor-receptor binding, mitogenesis, and angiogenesis. Cell. 1994;79:1005-1013. [Medline] [Order article via Infotrieve]
42.
Leonard CM, Bergman M, Frenz DA, Macreery LA, Newman
SA. Abnormal ambient glucose levels inhibit proteoglycan core
protein gene expression and reduce proteoglycan accumulation during
chondrogenesis: possible mechanism for teratogenic effects of maternal
diabetes. Proc Natl Acad Sci U S A. 1989;86:10113-10117.
43.
Rohrbach DH, Wagner CE, Star VL, Martin GR, Brown KS,
Yoon JW. Reduced synthesis of basement membrane heparan sulfate
proteoglycan in streptozotocin-induced diabetic mice.
J Biol Chem. 1983;258:11672-11677.
44. Moran A, Brown DM, Kim Y, Klein DJ. Effects of IGF-I and glucose on protein and proteoglycan synthesis by human fetal mesangial cells in culture. Diabetes. 1991;40:1346-1354. [Abstract]
45. Kaji T, Hiraga S, Yamamoto C, Sakamoto M, Nakashima Y, Sueishi K, Koizumi F. Tumor necrosis factor-alpha-induced alteration of glycosaminoglycans in cultured vascular smooth-muscle cells. Biochim Biophys Acta. 1993;1176:20-26.[Medline] [Order article via Infotrieve]
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