1 Hormones and the Vasculature Laboratory, 2 Cell Biology of Diabetes Laboratory, and 3 Molecular Signaling Laboratory, Baker Medical Research Institute, Melbourne, Victoria 8008, Australia
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ABSTRACT |
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We examined effects of
17-estradiol (E2) on human vascular smooth muscle cell
(VSMC) proliferation under normal (5 mmol/l) and high (25 mmol/l)
glucose concentrations. Platelet-derived growth factor (PDGF) BB (20 ng/ml)-induced increases in DNA synthesis and proliferation were
greater in high than normal glucose concentrations; the difference in
DNA synthesis was abolished by a protein kinase C (PKC)-
inhibitor,
LY-379196 (30 nmol/l). Western blotting showed that
PKC-
1 protein increased in cells exposed to high
glucose, whereas PKC-
protein and total PKC activity remained
unchanged, compared with normal glucose cultures. In normal glucose,
E2 (1-100 nmol/l) inhibited PDGF-induced DNA synthesis
by 18-37% and cell proliferation by 16-22% in a
concentration-dependent manner. The effects of E2 were
blocked by the estrogen receptor (ER) antagonist ICI-182780, indicating
ER dependence. In high glucose, the inhibitory effect of E2
on VSMC proliferation was abolished but was restored in the presence of
the PKC-
inhibitor LY-379196. Thus high glucose enhances human VSMC
proliferation and attenuates the antiproliferative effect of
E2 in VSMC via activation of PKC-
.
high glucose; estrogen; proliferation; smooth muscle cells; protein
kinase C-
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INTRODUCTION |
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IT IS CURRENTLY ACCEPTED that premenopausal women have relative protection from cardiovascular disease, compared with men and postmenopausal women, probably due to the protective effects of estrogen (7, 21). However, premenopausal women with diabetes mellitus lose this gender-based cardiovascular protection (3, 16), suggesting that hyperglycemia possibly overcomes some of the beneficial effects of sex steroids. To date, little is known of the cellular and subcellular interactions between the signaling pathways associated with hyperglycemia and sex hormones in the vasculature.
Several lines of evidence suggest that estrogens inhibit vascular
smooth muscle cell (VSMC) proliferation (14, 19, 23), whereas hyperglycemia stimulates VSMC growth (24, 26).
Because VSMC proliferation is an important cellular mechanism in the
development of atherosclerosis (17), an interaction
between estrogens and glucose-related signaling pathways in regulating
VSMC proliferation is possible. We hypothesized that high glucose
concentrations might attenuate the antiproliferative effect of
estrogens. In the present study, we examined the antiproliferative
effects of 17-estradiol (E2) under high (25 mmol/l
glucose) and normal (5 mmol/l glucose) glucose concentrations on human
internal mammary artery smooth muscle cells. We found that
E2 inhibits VSMC proliferation in a dose-dependent manner,
but its antiproliferative effect is abolished by high glucose
concentrations through a mechanism dependent on the activation of
protein kinase C (PKC)-
.
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METHODS |
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Cell culture.
VSMC were prepared from internal mammary arteries of two women with
coronary artery disease. The patients had hypertension and
hypercholesterolemia, but no diabetes, and one of the patients had had
a previous myocardial infarction. The internal mammary artery segments
were obtained at the time of coronary artery bypass grafting, and VSMC
were harvested by an explant technique (18). In brief,
segments of internal mammary artery were cut out and placed into
ice-cold Dulbecco's modified Eagle's medium (DMEM). All external fat
and connective tissue were cleaned from the vessel under microscope.
The outer membrane was torn off carefully, the vessel was cut
longitudinally, and the endothelial layer was removed by scraping with
forceps. Small strips of media were peeled off, transferred to 60-mm
dishes anchored under 9 × 22-mm sterile coverslips, and incubated
in normal glucose (5 mmol/l glucose) DMEM in the presence of
10% fetal bovine serum (FBS) in an incubator of 5% CO2 in
air at 37°C. Culture media were changed every three days, and after
~1 wk, VSMC were observed migrating from the pieces and growing on
the dishes. Cells were grown to near confluence and passaged, and the
presence of smooth muscle -actin was confirmed by immunofluorescence
staining and Western blot analysis as a marker of VSMC. Cells from the
first patient were used at passages 11-15, and cells from the
second patient were used at passages 6-7.
Estrogen receptor studies.
Estrogen receptor (ER) density in these VSMC was determined by
radioligand binding assay. Confluent monolayers of cells on 6-well
plates (~106 cells/well) were incubated with
[3H]estradiol (0.31-5 nmol/l) and nonradioactive
diethylstilbestrol (1 µmol/l) for 90 min. Cells were washed twice
with Dulbecco's PBS (D-PBS), scraped in the presence of 1.5 ml/well of
0.1% Triton-acetic acid, and put into scintillation vials with 5 ml of
Scintillation Liquid Instagel (Bio-Rad, Sydney, NSW, Australia)
for counting (5 min/vial) in a -counter. Functional ER studies were
carried out using the pure ER antagonist ICI-182780 (Tocris Cookson,
Bristol, UK).
Experimental protocol. Cells were seeded in culture plates, grown to ~90% confluence, and growth-arrested in serum-free DMEM with normal-glucose medium (5 mmol/l glucose, with 20 mmol/l mannose for control of osmolarity) or high- glucose medium (25 mmol/l glucose) for 48 h. Cell proliferation was induced with PDGF-BB (20 ng/ml), and effects of E2 were detected by preincubation with the hormone for 4 h before PDGF-BB stimulation.
Assays of cell proliferation.
DNA synthesis was determined by a [3H]thymidine
incorporation assay. Cells in 24-well plates were incubated with 1 µCi/well of [3H]thymidine during the last 3 h of
PDGF-BB treatment, washed three times with ice-cold D-PBS, incubated
with ice-cold 0.2 N HClO4 (1 ml/well) on ice for 30 min,
washed (0.5 ml/well, 3 times) with 0.2 N HClO4, incubated
with 0.5 ml/well of 0.2 N NaOH at 37°C for 1 h, and neutralized
with 0.2 ml/well of 6% acetic acid. Contents of the well were
transferred into scintillation vials with 3 ml of Instagel and counted
for 2 min per vial in a -counter. Cell number was measured by an
automatic cell counter (S.ST.II/ZM, Coulter Electronics, London,
UK) before and after PDGF-BB treatment for 48 h.
Analysis of PKC activity.
Cellular PKC activity was measured using a previously published method
(1). Briefly, cells in 24-well plates were stimulated with
PDGF-BB for 15 min and, after the medium was removed, incubated at
37°C for 10 min with 120 µl/well of assay buffer containing, in
mmol/l: 137 NaCl, 5.4 KCl, 0.3 Na2HPO4, 0.4 KH2PO4, 20 HEPES, 10 MgCl2, 5 EGTA,
25 -glycerophosphate, and 2.5 CaCl2 and 1 g/l glucose, pH 7.2-7.4, 50 mg/l digitonin, 0.05 mg/ml myristolated alanine-rich PKC kinase substrate (MARCKS) peptide, and 100 µmol/l [
-32P]ATP (added freshly before use). To
terminate the reaction, 30 µl/well of 25% trichloroacetic acid were
added for 5 min, and 135 µl of the cell lysate were transferred into
1.5-ml tubes containing 15 µl of 3.75% BSA (0.375 mg/ml final
concentration) and incubated on ice for 30 min. After being centrifuged
for 5 min at 12,000 g, 100 µl of supernatant were dotted
onto Whatman P-81 cation exchange paper (3 × 3 cm). After being
washed in 75 mmol/l phosphoric acid twice for 1 min, once for 5 min,
and three times for 10 min, the paper was dried and put into
scintillation vials containing Instagel (3 ml/vial) for counting (5 min) in a
-counter. Nonspecific background, defined as the amount of
radioactivity retained in the absence of PKC substrate MARCKS peptide,
was subtracted from all values. The PKC inhibitor LY-379196 was a gift
from Eli Lilly (Sydney, Australia). Similar to the compound LY-333531
(5, 25), LY-379196 at concentrations of 10-30 nmol/l
selectively inhibits PKC-
activity and at 600 nmol/l induces
nonselective PKC inhibition and can thus be used for analysis of total
and specific PKC activity.
Western blotting for protein expression of PKC subtypes.
Cells in 60-mm dishes were cultured under both normal- and high-glucose
conditions in the presence or absence of E2 (10 nmol/l) and
LY-379196 (30 nmol/l). Cells were lysed by incubation on ice for 30 min
with lysis buffer [in mmol/l: 20 Tris base, pH 7.7, 250 NaCl, 2 EDTA,
2 EGTA, 20 -glycerophosphate, and 1 Na-vanadate and 0.5% NP-40 and
10% glycerol; 10 µl/ml leupeptin, 5 µl/ml aprotinin, 1 µmol/l
pepstatin, 1 mmol/l 4-(2-aminoethyl)benzenesulfonyl fluoride, and 10 mmol/l dithiothreitol were added before use]. Plasma membrane proteins
were isolated by centrifugation at 14,000 rpm for 15 min, and 30 µg
of protein were electrophoresed on 10% SDS-polyacrylamide gels and
transferred to Hybond enhanced chemiluminescence (ECL) filters (Sigma).
The filters were blocked with 5% nonfat dry milk in TBST (20 mmol/l
Tris, pH7.5, 50 mmol/l NaCl, and 0.1% Tween-20) overnight and then
washed and incubated with primary antibodies against PKC-
or
PKC-
1 (Santa Cruz Biotechnology, Santa Cruz, CA) for
1 h. After being washed (3 × 10 min), blots were incubated with horseradish peroxidase-conjugated secondary antibody (DAKO) for
1 h, washed (3 × 10 min), incubated for 1 min with ECL
reagents (Amersham), and exposed to X-ray films. For protein loading
controls, the blots were washed again and probed with an anti-human
smooth
-actin antibody (DAKO) by use of the same method as
described. For quantification, bands were scanned in a PowerLook Scanner.
Statistical analysis. All data are presented as means ± SE. Comparisons between two means were made using Student's t-test and multiple comparisons using ANOVA. Differences of P < 0.05 were considered significant.
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RESULTS |
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E2 inhibits PDGF-BB-induced VSMC proliferation by a
mechanism dependent on ER and negatively regulated by high glucose
concentrations. In normal-glucose medium, preincubation of VSMC with
E2 (1-100 nmol/l) for 4 h resulted in a decrease
of PDGF-BB-induced DNA synthesis in a dose-dependent manner (Fig.
1), with inhibition reaching ~40% of
control at 10 and 100 nmol/l. In contrast, no inhibition was observed
for E2 in VSMC treated with PDGF-BB in high-glucose culture
cells (Fig. 1). Consistent with the effect on DNA synthesis, direct
cell counting showed a significant inhibition in PDGF-BB-stimulated
increase in cell number by E2 (1-10 nmol/l), an
inhibition observed for cells cultured in normal- but not in high-glucose medium (Fig. 2). High
glucose itself induced an increase in cell proliferation, and the
[3H]thymidine incorporation and cell number in high
glucose concentrations were higher than in normal glucose
concentrations (see controls in Figs. 1 and 2). Similar results were
also observed in VSMC from the aorta of Wistar Kyoto rats (data not
shown).
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Radioactive ligand binding assay showed that these human VSMC contained
estradiol-specific binding sites (ER) at a relatively high density
(24,000 ± 2,000 sites/cell; dissociation constant = 0.5 nmol/l, maximum binding capacity = 41 fmol), with no change in ER
density after 48-h culture in high-glucose medium (Fig. 3). In addition, the ER antagonist
ICI-182780 (100 nmol/l) completely abolished the inhibitory effect of
E2 (10 nmol/l) on cell DNA synthesis and proliferation
(Fig. 4), consistent with an ER-mediated effect.
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Involvement of PKC- in the stimulatory effect of high glucose on
VSMC proliferation.
Because PKC is a key regulatory element in signal transduction and
PKC-
has been implicated in diabetes-associated vascular complications (6, 12), we assessed the potential role of PKC in the effect of elevated glucose on E2 signaling by
determining the effects on PKC activity and protein expression and of
the PKC antagonist LY-379196.
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DISCUSSION |
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In the present study, we have shown that E2 inhibits
PDGF-BB-induced VSMC proliferation in a concentration-dependent manner but that this inhibition is lost in high-glucose cultures. We have
further shown that high-glucose medium induces an approximately threefold increase in PKC-1 protein expression after
24-h culture and that selective inhibition of PKC-
activity with
LY-379196 at 30 nmol/l inhibits high-glucose-induced cell proliferation and restores the antiproliferative effect of E2 in VSMC
cultured in high-glucose medium.
The effects of estrogens on the vasculature include nongenomic vasodilatation, reduction of circulating lipids (9, 22), and genomic actions on vascular cells (15). Some studies show that estrogens inhibit growth factor-induced cell proliferation in cultured VSMC via ER-mediated reduction of mitogen-activated protein (MAP) kinase (14), c-myc, and other early-response genes (15). It has also been shown, however, that cultured smooth muscle cells from human uterine artery or aortic smooth muscle cells from pregnant rats have higher proliferative responses to serum and PDGF and that E2 stimulates proliferation in these VSMC via activation of PKC (8). These studies indicate that estrogens have variable actions on VSMC, possibly dependent on the hormonal milieu of the cells, as well as their phenotype (20).
The present study shows that, in normal glucose, E2
attenuates PDGF-BB-induced proliferation in VSMC from human internal
mammary artery, similar to our previous observations showing that
E2 at physiological levels inhibits mechanical
strain-induced mitosis in human aortic smooth muscle cells
(11) and consistent with studies of cultured VSMC from
different sources (14, 19, 23). A salient finding of the
present study is that the antiproliferative effect of E2
was abolished under high glucose conditions via activation of PKC-.
Such an interpretation is supported by our observations that
high-glucose medium increased PKC-
1 protein expression
and selective inhibition of PKC-
(LY-379196 at 30 nmol/l) restored this antiproliferative effect of E2 in high-glucose conditions.
PDGF is an important factor in atherosclerosis. PDGF,
mainly as PDGF-BB, is produced and secreted by vascular
endothelial cells and contributes, via the PDGF- receptor, to VSMC
migration and proliferation (17). These PDGF actions on
VSMC are believed to be mediated through a complex array of
intracellular signaling pathways including MAP kinase, PKC, early
growth response genes, and intracellular calcium (4). The
antiproliferative effects of estrogens are reportedly mediated via the
MAP kinase pathway (2, 14). In the present study, total
PKC activity (Fig. 5) or PKC-
1 protein expression was
not affected by E2, indicating that the inhibitory effect
of E2 on PDGF-BB-induced proliferation is not via the PKC
pathway. The increased activation of PKC by high-glucose medium
counteracts the antiproliferative effect of E2 in VSMC. Our
results show that the antiproliferative effects of estrogen are
abolished by high-glucose medium but that this potentially beneficial
effect of estrogen can be restored, even in a high-glucose milieu, by a
PKC-
inhibitor.
Commonly used VSMC culture media such as DMEM and Waymouth's
medium contain a high level of glucose (25 mmol/l), much higher than
physiological levels (3-6 mmol/l). It has been shown, in the
present study and other studies (24, 26), that high
glucose itself induces VSMC growth, suggesting that glucose
concentrations in culture media possibly influence the results of
studies examining VSMC proliferation. The PKC pathway is regarded as a
major mechanism underlying the vascular effects of hyperglycemia.
Normalization of PKC by vitamin D (10) and of PKC- by
the selective inhibitor LY-333531 (1, 5) prevents the
vascular damage of hyperglycemia in experimental diabetes. Direct
effects of high glucose on cultured VSMC, as shown in the present study
and other studies (10, 24, 26), are also mainly via PKC
activation, especially the PKC-
isoform, which, possibly via
phospholipase D, induces vascular proliferation and hypertrophy, which
in turn likely contribute to diabetic vascular complications.
Our study provides a possible mechanism underlying the loss of
gender-based protection against vascular disease in diabetic women.
Vascular proliferation is a key element in diabetic macrovascular disease and is a significant determinant of morbidity and mortality in
this condition (13). Estrogen does not appear to inhibit growth factor-induced proliferation in the presence of high glucose concentrations. Nevertheless, our findings suggest that the
antiproliferative action of estrogen is restored by inhibition of
PKC- and may indicate a potential role for PKC-
inhibitors in
diabetic macrovascular disease in women.
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ACKNOWLEDGEMENTS |
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We are thankful to Dr. He Li for assistance in the PKC analysis, and Prof. J. Funder for critically reviewing the manuscript.
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FOOTNOTES |
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* K. Sudhir and P. A. Komesaroff have contributed equally to this study.
The study was supported by a block grant from the National Health and Medical Research Council (NH&MRC) of Australia to the Baker Institute. K. Sudhir is funded as a Senior Research Fellow of the NH&MRC. S. Ling is funded by Dora Lush Scholarship from the NH&MRC as a Ph.D. candidate in Monash University at Melbourne.
Address for reprint requests and other correspondence: Address for reprint requests and other correspondence: K. Sudhir, Pharmacyclics, 995 E. Arques Ave., Sunnyvale, CA 94085-4521 (E-mail: ksudhir{at}pcyc.com).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpendo.00111.2001
Received 9 March 2001; accepted in final form 14 November 2001.
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